System for Inhibiting Pathogenicity in the Rice-Blast Fungus Magnaporthe Grisea

ABSTRACT

In  Magnaporthe  species and other plant pathogenic fungal species, the appressoriu, (infection structure) is responsible for breaching the host plant cell wall and gaining entry into the host tissues.  Magnaporthe  ABC3 protein is an MDR transporter that plays an important role during host penetration and also is involved in regulating fungal response to intracellular oxidative stress. The insertional mutant abc3Δ, in which the ABC3 efflux pump function is blocked, lacks functional appressoria and is therefore incapable of causing disease in host plants. This invention provides the abc3 nucleic acid (gene) and ABC3 protein from  Magnaporthe  or from  Aspergillus, Ustilago  or  Fusarium  and describes methods for reducing plant pathogenicity for important rice pathogens.

This application claims the benefit of prior co-pending provisionalapplication Ser. No. 60/782,515, filed Mar. 16, 2006, the entirecontents of which are hereby incorporated by reference.

BACKGROUND OF THE INVENTION

1. Technical Field

This invention relates to the field of molecular biology and plantfungal disease. In particular, the invention relates to the discovery ofa fungal protein that is essential for pathogenicity in Magnaporthegrisea, a plant pathogen.

2. Description of the Background Art

The ascomycete Magnaporthe grisea causes disease in several monocotplant species, including blast disease in the important crop rice, andrepresents a model system to study fungal-plant interaction. See Ou etal., Rice Diseases, Commonwealth Mycological Institute, Surrey, 1985;Valent et al., “Rice blast as a model system for plant pathology”Phytopathology 80:33-36, 1990. Rice blast disease is an importantproblem in rice cultivation and a major threat to food security aroundthe world, causing very significant crop losses annually. M. grisea alsocan cause diseases in a number of other important cereal crops such aswheat, rye and barley, and also in weed and turf grasses.

The asexual life cycle of M. grisea involves sporulation of the fungalhyphae to produce fruiting structures called conidia. The conidiacontain many spores, which germinate in a suitable environment on a hostplant, and develop to form a specialized infection structure termed anappressorium. These appressoria penetrate the plant cell by producing apenetration peg that is forced through the plant cell wall to begininfection. Upon entry of the fungus into the host cells, the fungusproliferates by forming penetration and infectious hyphae that establishand spread the disease lesions. See Hamer and Talbot, “Infection-relateddevelopment in the rice blast fungus Magnaporthe grisea.” Curr. Opin.Microbiol., 1:693-697, 1998; Talbot, “On the trail of a cereal killer:Exploring the biology of Magnaporthe grisea” Annu. Rev. Microbiol.57:177-202, 2003. Infection of immature plants often is fatal. Whilemature plants usually survive, crop yield is considerably curtailed dueto reduced photosynthesis and use of photosynthate by the invadingfungal hyphae.

Currently, outbreaks of blast disease are controlled by applyingexpensive and toxic fungicidal chemicals such as probenazole,tricyclazole, pyroquilon and phthalide, or by burning infected crops.These methods are only partially successful since the fungus can developresistance to the chemical agents and weed grasses may serve as adisease reservoir after burning. Clearly, improvements are needed inmethods to control blast disease in rice and other cereal crops.

Transmembrane proteins belonging to the ubiquitous ATP-binding cassette(ABC) superfamily have been identified in several genera of prokaryotesand eukaryotes, including fungi. See Higgins, “ABC transporters: frommicroorganisms to man” Annu. Rev. Cell Biol. 8:67-113, 1992. TheP-glycoproteins are a subfamily of plasma membrane-localized ABCtransporters that modulate a multi-drug resistance (MDR)-related effluxof a broad range of compounds such as sugars, inorganic ions, heavymetal ions, peptides, lipids, metabolic poisons and drugs to effectivelyreduce the cellular accumulation of toxic compounds by efflux from theinner leaflet of the plasma membrane to the cell exterior. SeeKolaczkowski et al., “In vivo characterization of the drug resistanceprofile of the major ABC transporters and other components of the yeastpleiotropic drug resistance network” Microb. Drug Resist. 4:143-158,1998; Driessen et al., “Diversity of transport mechanisms: commonstructural principles” Trends Biochem. Sci., 25:397-401, 2000. ABCtransporters play a major role in multidrug resistance (MDR), amechanism that operates in mammalian tumor cells. The MDR subfamily ofefflux pumps includes the multidrug resistance P-glycoproteins. SeveralMDR-type P-glycoproteins have been shown to catalyze the ATP-dependentefflux of anti-tumor agents during cancer chemotherapy. See Cole et al.,“Overexpression of a transporter gene in a multidrug-resistant humanlung cancer cell line” Science, 258:1650-1654, 1992; Gottesman andPastan, “Biochemistry of multidrug resistance mediated by the multidrugtransporter” Annu. Rev. Biochem., 62:385-427, 1993.

Identification and subsequent sequence comparisons of more than ahundred genes encoding ABC transporters show that the ABC proteins haveone or two well-conserved nucleotide-binding folds of approximately 200amino acid residues and the Walker A and B motifs as well as the SGG(Q)signature. Michaelis and Berkower, “Sequence comparison of yeastATP-binding cassette proteins” Cold Spring Harbor Symp., Quant. Biol.LX:291-307, 1995. Several members of this large superfamily transportcytotoxic agents across biological membranes, reducing the intracellularlevel of toxins and metabolites. Driessen et al., “Diversity oftransport mechanisms: common structural principles” Trends Biochem. Sci.25:397-401, 2000.

In filamentous fungi, ABC transporter activity likely is involved inenergy-dependent efflux of fungicides or phytoalexins in Aspergillusnidulans (Andrade et al., “The ABC transporter AtrB from Aspergillusnidulans mediates resistance to all major classes of fungicides and somenatural toxic compounds” Microbiology, 146:1987-1997, 2000; Andrade etal., “The role of ABC transporters from Aspergillus nidulans inprotection against cytotoxic agents and in antibiotic production” Mol.Gen. Genet., 263:966-977, 2000), A. fumigatus (Tobin et al., “Genesencoding multiple drug resistance-like proteins in Aspergillus fumigatusand Aspergillus flavus” Gene, 200:11-23, 1997), Botrytis cinerea(Vermeulen et al., “The ABC transporter BcatrB from Botrytis cinerea isa determinant of the activity of the phenylpyrrole fungicidefludioxonil” Pest Manag. Sci. 57:393-402, 2001), M. grisea (Urban etal., “An ATP-driven efflux pump is a novel pathogenicity factor in riceblast disease” EMBO J. 18:512-521, 1999), Gibberella pulicaris(Fleissner et al., “An ATP-binding cassette multidrug-resistancetransporter is necessary for tolerance of Gibberella pulicaris tophytoalexins and virulence on potato tubers” Mol. Plant-MicrobeInteract. 15: 102-108, 2002) and Mycosphaerella graminicola(Stergiopoulos et al., “The ABC transporter MgAtr4 is a virulence factorof Mycosphaerella graminicola that affects colonization of substomatalcavities in wheat leaves” Mol. Plant Microbe Interact. 16:689-698, 2003;Zwiers et al., “ABC transporters of the wheat pathogen Mycosphaerellagraminicola function as protectants against biotic and xenobiotic toxiccompounds” Mol. Genet. Genomics 269:499-507, 2003). However, none ofthese previously known fungal transporter molecules belong to theP-glycoprotein or MDR subfamily of the ABC transporters.

SUMMARY OF THE INVENTION

Embodiments of the invention described and claimed herein include anabc3 gene of SEQ ID NO:1 and an ABC3 protein of SEQ ID NO:2. Theinvention's embodiments also include an isolated nucleic acid encodingthe protein-coding region of the abc3 gene, which can be selected fromthose of Magnaporthe, Aspergillus, Ustilago or Fusarium species abc3gene, for example.

An embodiment of the invention is the abc3 gene, which preferably is SEQID NO:1. A further embodiment of the invention is an ABC3 protein, whichpreferably is SEQ ID NO:2. Included in the invention are nucleic acidsthat encode an ABC3 protein as listed above and described herein.

Further embodiments of the invention include a method of rendering plantpathogenic species including Magnaporthe species, such as Magnaporthegrisea, or Aspergillus, Ustilago or Fusarium species, non-pathogenic toa plant by applying an ABC3 inhibitor to the plant or expressing an ABC3inhibitor in the plant. ABC3 inhibitors may be selected from a peptide,a nucleic acid that encodes the peptide, a protein, a nucleic acid thatencodes the protein, a peptide-mimetic compound, an organic chemical, areplacement plasmid vector, an antisense nucleic acid or any combinationthereof. One example of an ABC3 inhibitor is pFGLabcKO.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a phylogram of MDR proteins related to Abc3 protein, depictingthe phylogenetic relatedness of Magnaporthe ABC1 and ABC1 proteins withthe MDR or ABC proteins from the yeasts Saccharomyces (ScSTE6 andScPDR5) Schizosaccharomyces (SpPMD1), and Candida (CaCDR1). Theevolutionary distance scale (Mya) and the branch lengths are indicated.Bootstrapping (1000 replicates/iterations) was used to generate thephylogenetic tree using the MEGA 3.1 Neighbor-joining algorithm.

FIG. 2 is a phylogram of MDR proteins related to Abc3 protein, depictingthe most parsimonious phylogenetic relatedness of Magnaporthe ABC3protein with the MDR proteins from the indicated genera withineukaryotes.

FIG. 3 is a multiple sequence alignment used for the phylogeneticanalysis of ABC3-related MDR proteins. Red indicates identical aminoacids; blue indicates similar amino acid residues. Dashed lines or dotsindicate gaps in the alignment. MgABC3 (Magnaporthe grisea)=SEQ IDNO:25; ScSTE6 (Saccharomyces ceriviseae)=SEQ ID NO:26; SpPMD1(Schizosaccharomyces pombe)=SEQ ID NO:27; ScPDR5 (Saccharomycesceriviseae)=SEQ ID NO:28; CaCDR1 (Candida albicans)=SEQ ID NO:29; MgABC1(Magnaporthe grisea)=SEQ ID NO:30.

FIGS. 4A and 4B are photographs of barley leaf explants inoculated withconidiospores of TMT2807 or wild-type (WT) strain.

FIGS. 5A and 5B are photographs of barley leaf explants inoculated withconidiospores of TMT2807 or wild-type (WT) strain.

FIG. 6 is a schematic representation of the annotated ABC3 locusspanning a HindIII-PvuII fragment from Contig 67 on Supercontig 5.117 inM. grisea.

FIGS. 7A and 7B show Southern blot analyses of the ABC3 mutants and theComplemented strain.

FIG. 8 shows the gene sequence of abc3 (SEQ ID NO:1). Exons are shown inupper case letters; introns are shown in lower case letters.

FIG. 9 shows the protein sequence of ABC3 (SEQ ID NO:2).

FIG. 10 is a photograph of a wild-type, an abc3Δ mutant and an abc3Δstrain complemented with the full length ABC3 (Comp). The strains weregrown on complete medium for a week and photographed. Scale bar=1 cm.

FIG. 11 is a bar graph showing perithecia development by the indicatedMagnaporthe strains in individual sexual crosses with the tester strainof the opposite mating type, assessed 3 weeks post inoculation.

FIG. 12 is a photograph of conidia from the wild type or abc3Δ strainwhich were allowed to germinate on complete medium for 16 hours and thenvisualized after acid fuchsin staining. Bar=20 microns.

FIGS. 13A and 13B are photographs showing the morphology of appressoria24 hours after inoculation on a hydrophobic inductive surface. Bar=10mm.

FIG. 14 shows hypersensitivity-reaction (HR) assessed 48 hours afterinfection with Magnaporthe strains and represent the ratio of rice Pr-1gene expression to that of the Actin transcript. Bar=1 cm.

FIG. 15 shows barley leaves challenged with wild type or abc3Δ conidiaand co-stained with DAB and Trypan blue to detect peroxide- andHR-associated cell death, respectively. The arrowhead indicates a rareHR event in the host upon challenge with the mutant.

FIG. 16 shows conidia from wild type or abc3Δ mutant inoculated onabraded barley leaves (FIG. 17, upper panels) or injected at the base ofbarley leaves (FIG. 17, central panels) or the base of rice-leaf sheaths(FIG. 17, bottom panels). Bar 0.5 cm.

FIG. 17 shows equivalent numbers of conidia from wild type or abc3Δmutant were inoculated onto synthetic membranes (PUDO-193 cellophane)and assessed for germination, appressoria formation, surface penetrationand invasive growth. The arrowhead indicates invasive growth. Bar=10 mm.

FIG. 18 is a photograph of penetration and invasion 48 hours afterinoculation on onion epidermal strips. Bar=10 mm.

FIG. 19 is a photograph of penetration and invasion 48 hours afterinoculation on barley leaf explants. Bar=10 mm.

FIG. 20 shows data for appressorium formation at 24 hours andappressorium function (host penetration and invasion, as judged byaniline blue staining of resultant callose deposits) at 48 hours (wildtype, shaded; abc3Δ mutant, black).

FIG. 21 is a set of transmission electron photomicrographs showingbarley leaves challenged with conidia from wild type or abc3Δ mutant atthe indicated time points. Bar=2 mm, except for the middle right panelwhere it denotes 1 mm.

FIG. 22 is a pair of photographs showing wild type or abc3Δ mutantconidia, inoculated on Barley-leaf explants, onion epidermal strips orrice-leaf sheaths and processed for aniline blue staining after 72hours.

FIG. 23 is a bar graph presenting quantified data from FIGS. 21 and 22.

FIG. 24 shows the results of an appressorial assay performed in a hostor on cellophane, comparing viability and invasive growth of wild typeand abc3Δ mutant conidia.

FIG. 25 is a photograph showing serial dilutions of wild type or abc3Δconidial suspensions, cultured in the presence (20 mg/ml) or absence (0mg/ml) of Valinomycin on complete growth medium for 5 days.

FIG. 26 is a photograph of wild type or abc3Δ conidial suspensions,cultured in the presence (20 mg/ml) or absence (0 mg/ml) of Valinomycinon artificial inductive membranes for 36 hours. Bar=15 mm.

FIG. 27 shows fission yeast strains as indicated after 5 days growth onYES medium in the presence or absence of Valinomycin.

FIG. 28 shows mycelial plugs from the wild type or abc3Δ strain culturedon minimal agar medium with the indicated amounts of hydrogen peroxide.

FIG. 29 shows serial dilutions of conidiospore suspensions from the wildtype or abc3Δ strain cultured on minimal agar medium with the indicatedamounts of hydrogen peroxide.

FIG. 30 shows three-(top panels) or six-(bottom panels) day-old coloniesof wild type or abc3Δ colonies, stained with NBT solution to detectperoxide accumulation.

FIG. 31 shows wild type and abc3Δ appressoria formed on artificialmembranes (upper panels) or on barley leaf explants (lower panels),stained with diaminobenzidine to detect the accumulation of hydrogenperoxide (arrowheads) Bar=10 mm.

FIG. 32 is a fluorometric analysis showing total extracts from wild typeor abc3Δ appressoria, treated with CM-DCFDA.

FIG. 33 is a bar graph showing the degree of restoration of appressoriumfunction by antioxidant treatment. Values in FIG. 33 represent mean±SDfrom three independent experiments each involving 2000 conidia perstrain.

FIG. 34 shows the percentage of appressoria that elaborated penetrationpegs in the presence and absence of 3 mM peroxide.

FIG. 35 is a set of photographs of mycelia and conidia harvested from astrain expressing an ABC3-GFP fusion protein and imaged usinglaser-scanning confocal microscopy to detect the eGFP signal. Bar=10 mm.

FIG. 36 is a set of photographs showing appressorium development in thegerm tubes of a Magnaporthe strain expressing ABC3-GFP strain at theindicated time points, imaged using GFP epifluorescence. Bar=10 mm.

FIG. 37 is a set of photographs showing vacuolar distribution ofABC3-GFP. Scale bars denote 10 mm.

FIG. 38 is a set of photographs showing confocal microscopy assistedanalysis of ABC3-GFP localization in the penetration structures(arrowhead) elaborated by the appressoria in PUDO-193 membrane. Bar=10micron.

FIG. 39 shows appressorium development over the indicated time course,imaged using GFP epifluorescence. The bar equals 10 mm. Arrowheadsindicate the vacuolar distribution of ABC3-GFP.

FIG. 40 is a set of photographs showing the effect of intracellular andextracellular extracts prepared from the appressoria of the wild type orabc3Δ strain in barley leaf infection assays.

FIG. 41 is a bar graph showing appressorium function in the absence orpresence of the intracellular neutral extract from the abc3Δ mutant.Values represent mean±SD from three replicates of the experiments.

FIG. 42 is a bar graph showing cell viability assessed with Phloxine Bstaining in vegetative mycelia from the wild type (gray) or abc3Δ(black) strains.

FIG. 43 shows the results of barley-leaf inoculation assays in theabsence (−EXT; solvent control) or presence (+EXT; at 1× or 0.5×concentration) with wild type conidia.

FIG. 44 is a set of epifluorescent photomicrographs showing conidia fromthe ABC3(p)::deGFP strain which were allowed to undergo appressoriumdevelopment for 24 hours in the absence (Control) or presence (Treated)of 1 mM hydrogen peroxide. Bar=10 mm.

FIG. 45 shows results of semi-quantitative RT-PCR amplification usingABC3 or MgCHAP1 or MgTUB1 specific primers from total RNA extracted fromthe wild type strain grown for the indicated hours post inoculation(hpi) on barley leaves.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

To successfully invade a host plant and to cause blast disease,phytopathogenic fungi such as M. grisea need to adapt to the specificenvironment of the host and overcome or survive the cytotoxic andantifungal compounds or phytoalexins produced by the plant hosts. SeeDixon et al., “Early events in the activation of plant defenseresponses.” Annu. Rev. Phytopathol. 32:479-501, 1994; Kodama et al.,“Sakuranetin, a flavonone phytoalexin from ultraviolet-irradiated riceleaves.” Phytochemistry 31:3807-3809, 1992; Osbourn, “PreformedAntimicrobial Compounds and Plant Defense against Fungal Attack” PlantCell 8:1821-1831, 1996. Intragenome BLAST (Magnaporthe Genome Database,Broad Institute, USA) computer searches revealed that the Magnaporthegenome encodes at least 76 ABC-like transporters.

The gene discovered here was found to be unique and showed scantsimilarity to the other ABC molecules within the Magnaporthe genome andproteome, and thus failed to predict any true orthologs. The datapresented here showed that ABC3 protein is a transmembrane proteinenriched in the outer membranes of the infection structure. Asignificant distribution of ABC3 protein also was observed in thevacuoles during the late stages of appressorium formation. Hostinfection data clearly showed that ABC3 protein has acquired a morespecialized role in pathogenicity during its evolution from or alongwith the yeast orthologs.

TMT2807 was isolated as a non-pathogenic mutant in Magnaporthe.Molecular genetics and further characterization of TMT2807 enabledidentification of the MDR P-glycoprotein encoding gene abc3 as apathogenicity factor in Magnaporthe. To confirm the role of ABC3 proteinin Magnaporthe pathogenesis, abc3Δ mutants were created andcharacterized in two separate wild type backgrounds (Guy11 and B157).The studies clearly demonstrated that loss of ABC3 protein inMagnaporthe completely destroys pathogenicity towards rice and barley.The abc3Δ colonies were slow growing compared to the corresponding wildtype, but were largely unaffected in morphology and in conidiaformation. Genetic complementation analyses confirmed that the defectsseen in the TMT2807 and abc3Δ strains were due solely to the loss ofABC3 function.

Mutational analysis of the gene abc3, has allowed definition of animportant role for MDR-based efflux during the host penetration step ofMagnaporthe pathogenesis. Mutants lacking the abc3 gene (abc3Δ) werecompletely nonpathogenic, but surprisingly were capable of penetratingthin cellophane membranes to a limited extent. Studies described belowshowed that the inability of abc3Δ to penetrate the host surface is mostlikely due to excessive buildup of peroxide and accumulation ofinhibitory metabolite(s) within the mutant appressoria. Treatment withantioxidants partially suppressed the host penetration defects in theabc3Δ mutant. The amount of the inhibitory metabolite(s) and/or thetiming of their production may be the limiting factors that allow themutant to escape the maximal activity of these inhibitors on cellophane.Another likely possibility is that maximum turgor generation is probablynot necessary on cellophane surfaces.

ABC3 protein is required for Magnaporthe pathogenesis. Its function ismost likely critical at the host penetration step during blast diseaseestablishment. The vast majority (˜99%) of abc3Δ appressoria lackingABC3 function failed to breach the host surface even upon extendedincubation. Using papillary callose deposits and TEM analyses, it wasdiscovered that only a negligible number of mutant appressoria madefutile attempts at host penetration, however the fungus was able toelicit weak hypersensitivity reaction within the host tissue duringcompatible and incompatible interactions, as well as upon hostinoculation through wounds or by injection.

The abc3Δ mutant was highly sensitive to oxidative stress and was unableto survive the host environment and invasive growth conditions. The ABC3transcript was upregulated at the early stages of plant infection.

The studies reported here have determined that functional ABC3 isrequired for proper operation of appressoria (infection structures)during the initial pathogenic phase of rice-blast fungus. Without ABC3function, host penetration during the disease establishment phase doesnot take place. As a consequence, mutants lacking operational ABC3 arecompletely nonpathogenic. Fungal mycelia and appressoria from theABC3-deleted strain showed a multidrug-sensitive phenotype when testedwith several different classes of peptide antibiotics such asvalinomycin. This suggests that ABC3 protein is required for the effluxof these or other toxic compounds across the fungal membrane undernormal circumstances.

ABC3 protein transcript levels were redox-responsive, and on hostsurfaces, activation of ABC3 occurred during initial stages of blastdisease establishment. Model compounds of oxygen radical production,such as paraquat and hydrogen peroxide, modulated ABC3 protein levels ina redox-sensitive manner. Cells lacking ABC3 protein were sensitive tooxidative stress but not to osmotic shock or nutrient deprivation. Thissuggests that the loss of appressorium function in abc3Δ mutants is aconsequence of the mutant's inability to cope with cytotoxic andoxidative stress. The data presented here therefore indicate that ABC3function helps Magnaporthe to cope with cytotoxicity and oxidativestress within the appressoria during early stages of infection-relatedmorphogenesis and that it likely imparts a defense against certainantagonistic and xenobiotic conditions encountered during pathogenicdevelopment.

Thus, ABC3 function provides the blast fungus with an important defenseagainst the xenobiotic and antagonistic environment encountered on andin the host plant. Taking advantage of this mechanism, embodiments ofthe invention provide a method of combatting blast diseases caused by M.grisea or any other fungus expressing ABC3 protein by inhibiting theprotein function in the plant or plant environment. This may beaccomplished by applying ABC3 inhibitors to the plant or the environmentaround the plant or by causing the plant to express inhibitors.

Exogenously applied intracellular extracts from abc3Δ appressoriareduced the wild type strain's ability to penetrate host surfaces andcellophane membranes. This suggests that the abc3Δ mutant is defectivein toxic metabolite efflux and that ABC3 transporter activity is mostprobably required for the timely efflux of certain inhibitorymetabolite(s) during the host penetration step of blast infections. Theaccumulation of these toxic metabolites in appressoria likely causespenetration inhibition. Exogenous addition of the toxic metabolite(s)during wild type infection assays discernibly reduced blast symptoms,but hyper-sensitivity reaction was noticeable in the host leaf even inthe absence of the blast fungus.

Cytorrhysis assays (Howard et al., “Penetration of hand substrates by afungus employing enormous turgor pressures” Proc. Natl. Acad. Sci. USA,88:11281-11284, 1991; de Jong et al., “Glycerol generates turgorpressure in rice blast” Nature, 389:244-245, 1997) revealed that therewas no significant reduction in the overall turgor generated by abc3Δappressoria, despite having an average size slightly larger than thewild type appressoria. The abc3Δ appressoria were capable of penetratingcellophane membranes, albeit less efficiently than the wild type strain.Subsequent invasive growth and spread within the cellophane membraneswere however significantly compromised in the abc3Δ mutant, which alsoshowed reduced viability under these conditions.

To begin this study, a forward genetics approach was used to isolate amutant, TMT2807, of the rice-blast fungus Magnaporthe grisea. TMT2807was identified as a mutant incapable of causing blast disease on barleyleaves although its infection structure formation appeared normal.Molecular genetics and further characterization of this mutant strainrevealed that M. grisea contains a P-glycoprotein-encoding gene,hereinafter termed abc3, and that loss of function of this gene resultedin loss of pathogenicity.

With Agrobacterium T-DNA-mediated insertional mutagenesis, ABC3 functionwas identified as an essential factor in host penetration duringMagnaporthe pathogenicity. Further characterization revealed that theABC3 protein functions during the late appressorium stage and isrequired for successful entry of the fungus into host tissue. Therefore,loss of ABC3 protein results in cessation of infection-relatedmorphogenesis which occurs during plant host penetration by the fungus.As a consequence, mutants lacking ABC3 were incapable of hostpenetration. The abc3 gene therefore is a new virulence determinant inMagnaporthe.

The role of ABC3 in Magnaporthe pathogenesis was confirmed by studies onTMT2807 and a Magnaporthe insertional mutant (abc3Δ) created in twoseparate wild type backgrounds which lack the P-glycoprotein-basedefflux function. DNA fragments (about 1.1 kb each) representing the 5′and 3′ UTR of ABC3 gene were obtained by PCR, ligated sequentially so asto flank the HPH1 cassette in pFGL44 to obtain plasmid vector pFGLabcKO.Linearized pFGLabcKO was introduced into wild-type M. grisea for aone-step replacement of the ABC3 gene with HPH1 cassette. The completeM. grisea ABC3 locus was obtained as a 6.3 kb HindIII fragment from theBAC 22C21 and cloned into the HindIII site of pFGL97 to obtain pFGLr2with resistance to bialaphos or ammonium gluphosinate as a fungalselectable marker for the rescue transformation. See Example 1. Southernblots analyses were performed to confirm successful gene deletionstrains and single-copy genomic integration of the ABC3 rescuingcassette. Flanking sequence analysis suggested that the insertionoccurred in an as yet un-annotated region represented by Contig 2.904from the Magnaporthe genome sequencing project. There also was adiscrepancy in the neighboring contigs (contigs 2.903 and contig 2.902do not exist). The disrupted gene locus of these mutants (abc3) encodesa protein with extensive homology to the Cluster II.2 (TC#3.A.1.201)type P-glycoprotein transporters such as human MDR1.

Annotation of the coding region confirmed this. A targeted gene deletionexercise for the complete open reading frame of the annotated gene(abc3) was performed to assess its role in Magnaporthe pathogenicity.Mycelia from the resulting mutant abc3Δ strain showed a slight reductionin vegetative growth on rich medium, although conidia formation remainedunaffected. This suggested that ABC3 function is needed for maximalvegetative growth of Magnaporthe. Loss of ABC3 function, however,completely abolished appressorial function, which led to a completecessation of host penetration. Complementation analysis using afull-length abc3 genomic clone rescued all the phenotypic defectsassociated with the loss of ABC3 function.

ABC3 protein showed extensive similarity to several MDR-like proteinsfrom filamentous fungi, most notably Phaeosphaeria, Fusarium, Neurosporaand Aspergillus species. Intragenome BLAST (Magnaporthe Genome Database,Broad Institute, USA) searches using ABC3 protein as the query revealedthat Magnaporthe genome encodes at least 76 ABC-like transporters, andfurther identified a paralog (MG09931.4; 56% similarity) within theMagnaporthe proteome. Such paralogs also were uncovered in Aspergillus,Fusarium and Neurospora species. Their occurrence suggested a recentgene duplication event.

Phylogenetic analyses revealed that ABC3 protein most likely belongs toa distinct class of MDR transporters that is separate from thoserepresented by Pmd1 and AtrC. ABC3 protein from M. grisea had extensivesimilarity to the mammalian (human) MDR/P-glycoprotein, HsMDR1, and toseveral MDR-like proteins from fungi and yeasts. It was most closelyrelated to Fg6881, a hypothetical ORF from Fusarium spp.

ABC3 protein was able to functionally replace Pmd1 (its potentialortholog from the fission yeast) and, like Pmd1, regulated resistancetowards cyclic peptide antibiotics such as Valinomycin. Compared to wildtype fission yeast cells, however, the pmd1A mutant did not show anygrowth-related defects in contact with barley or rice leaf surfaces.Unlike the abc3-related Step 6 in budding yeast, Pmd1 is not requiredfor mating. However, abc3 seems to have retained at least some importantfunction in the mating pathway since loss of ABC3 led to a decrease inthe overall number of perithecia. ABC3 protein, like its ortholog Step 6from budding yeast, is required for fertility of the blast fungus. Theclosely related AfuMDR1 from A. fumigatus is specific for the antifungalagent Cilofungin, whereas AtrD, which is 76% identical to AfuMDR1,serves as an efflux pump for fenarimol. Thus the degree of primarysequence homology does not correlate strictly with similar substratespecificity.

The inability to recover viable cultures of the abc3Δ mutant from theinoculation sites in the host beyond 30 hours post infection alsoindicates that ABC3 protein is likely important for in planta survivaland spread of Magnaporthe. In preliminary analyses the abc3Δ mutant'sloss of viability appeared to be independent of the autophagic celldeath pathway that has recently been implicated in conidial cell deathduring initiation of blast disease. Alternatively, it is possible thatthe increased cell death in the mutant is a consequence of excessive ROSaccumulation.

The function of ABC3 protein in the regulation of oxidative stress isdifferent from that found in its counterparts in fungus. The resultspresented here showed that loss of ABC3 protein renders the blast fungusmore sensitive to oxidative damage. Compared to the wild type, abc3Δmutant appressoria accumulate significantly higher levels of reactiveoxygen intermediates. This peroxide accretion occurred under in vitroand in planta conditions. Suppressing this peroxide accumulation withexogenous antioxidants partially suppressed the host penetration defectsin the abc3Δ mutant. Conversely, addition of exogenous peroxide,menadione or paraquat during infection assays significantly blocked thegrowth and function of wild type Magnaporthe conidia and appressoria.These data taken together indicate that the failure of abc3Δ appressoriato invade the host is caused by the cumulative effect of: (1) theaccumulation of inhibitory metabolite(s) and (2) an excessive build-upof reactive oxygen intermediates. An observation of interest was theredox-sensitive regulation of ABC3 expression demonstrated using a GFPreporter construct. This suggests that ABC3 protein is a new MDR orP-glycoprotein that regulates fungal pathogenicity per se as well as theability of the blast fungus to withstand host-specific environmentalconditions.

Magnaporthe host plants are known to secrete a number of phytotoxins andother pathogenesis-related proteins upon fungal challenge. See, forexample, Dixon et al., “Early events in the activation of plant defenseresponses” Annu. Rev. Phytopathol., 32:479-501, 1994; Osbourn,“Preformed Antimicrobial Compounds and Plant Defense against FungalAttack” Plant Cell, 8:1821-1831, 1996; Kodama et al., “Sakuranetin, aflavonone phytoalexin from ultraviolet-irradiated rice leaves”Phytochemistry, 31:3807-3809, 1992. It is therefore likely that theabc3Δ mutant accumulates intracellularly toxic antifungal agent(s) ofplant origin and that it is this which ultimately halts fungal growthand proliferation. The same would be true of any blast fungus thatlacked sufficient ABC3 protein function, and therefore can serve as apoint to combat blast disease. Alternatively, the abc3Δ mutant might beincapable of transporting as yet unidentified pathway intermediate(s) orhost-specific toxic compound(s) that have deleterious effects within thefungal appressoria. The abc3Δ mutants lacking ABC3 protein function herefailed to penetrate the host surface even upon extended incubation.Interestingly, these mutants were histologically similar to wild typewhen grown on artificial surfaces and were fully capable of invasivegrowth and spread in these surfaces. Thus, it appears likely that theABC3 transporter is required for the efflux of one or more host-specificcompounds during the penetration step.

Localization studies using an ABC3:GFP fusion protein showed that ABC3is a plasma membrane resident protein in early appressoria and inpenetration hyphae and thus would be accessible to peptide or similarantifungal agents in the external environment. Interestingly, ABC3protein also localizes to the vacuolar lumen during the lateappressorial stage, thus making it available to antifungal-agentsaccessible to the internal membrane compartments. Subsequently, theABC3-GFP became plasma membrane-associated in primary penetrationhyphae.

Thus, ABC3 is an important pathogenicity factor in Magnaporthe (andorthologs available in all pathogenic fungi sequenced thus far) that canbe controlled using antifungal agents or inhibitors directed against it.Because ABC3 is a fungal-membrane resident pump involved in efflux ofone or more toxic metabolites or inhibitors of appressoria function,deletion of this efflux function leads to hypersensitivity to hydrogenperoxide levels in the abc3Δ strain. This mechanism could play a role inregulation either of peroxide levels or intermediates derived fromperoxide-related metabolism. The ABC3 protein also could function as acounter-defense mechanism in down-regulating the host response, based onthe build-up of reactive oxygen species. Identifying molecules upstreamand downstream of ABC3 and its potential substrates could lead toimportant molecular insights into the signaling mechanisms that areessential to host penetration by the fungus.

Inhibitors of ABC3 protein can be applied to plants or to theenvironment to impair or otherwise modify ABC3 function and block hostpenetration by the blast fungus and thus disease in the plant host. SuchABC3 inhibitors may include peptide or peptide-mimetic compounds,proteins or nucleic acids, for example a regulatory element, anantisense molecule or a replacement plasmid vector that knocks out theabc3 gene, which interfere with ABC3 function or expression. Peptide orprotein inhibitors may be applied to the plant to be treated orprotected. Small molecule (i.e. drug-like organic chemical molecules andthe like as are known in the art) inhibitors of ABC3 protein functionalso are contemplated for use in methods of treating or preventing blastdisease. Such small molecule inhibitors or peptide inhibitors may beidentified according to conventional screening methods known in the art.Small molecule fungicides that specifically inhibit ABC3 function can besprayed directly on the host plants to reduce fungal penetration anddisease without the broad toxicity caused by traditional anti-fungalagents. Thus, an ABC3 inhibitor is any molecule which, when present inthe plant, on the plant or in contact with the Magnaporthe pathogen.

Alternatively, host plants can be engineered to express protein orpeptide ABC3 inhibitors, to create disease-resistant plants. The plantmay be transformed with a gene such that the protein or peptideinhibitor is expressed in the plant or in appropriate tissues of theplant. Methods for producing transgenic plants of this type are known inthe art, and any conventional methods are contemplated for use with thisinvention. Because other fungal species, including other pathogens,express related multi-drug resistance pumps, these methods would beuseful in modifying the activity of ABC3 homologs in other fungalpathogens to reduce and control their ability to cause disease as well.

The fungal protein ABC3 therefore could be used in designing effectivecontrol agents against blast disease per se. For example, small moleculeinhibitors or short peptides that block the function of ABC3 protein orperturb the requisite metabolic pathway(s) could be designed and suchinhibitors would be, for example, spray inoculated or expressed inplanta to prevent establishment and/or spread of disease. Suchinhibiting agents also can be used on ABC3 orthologs in other pathogenicfungi to control the respective fungal diseases of plants and animals.Such orthologs include the abc3-encoding nucleic acids and ABC3 proteinsof any plant pathogenic Aspergillus, Ustilago and Fusarium species aswell as Magnaporthe species. These orthologs therefore are consideredpart of this invention, as well as methods using their analogous tothose disclosed herein.

EXAMPLES Example 1 Identification and Isolation of the ABC3 Locus

Schizosaccharomyces pombe wild-type strain YNB38 (h-,ade6-M216/leu1-32/ura4-D18/his3-D1) was maintained on yeast-extract plussupplements (YES) agar medium or under selection on Edinburgh minimalmedia (EMM), with appropriate supplements, using standard techniques asdescribed in Moreno et al., “Molecular genetic analysis of fission yeastSchizosaccharomyces pombe” Meth. Enzymol., 194:795-823, 1991. Strainswere grown on agar plates or in liquid media at 32° C.

Wild-type strains of M. grisea, Guy11 (mat1-2) and TH3 (mat1-1), wereused in the following methods. Guy11, and the abc3Δ knockout andcomplementation strains were cultured on prune-agar medium or completemedium (CM) as described by Soundararajan et al., “Woronin body functionin Magnaporthe grisea is essential for efficient pathogenesis and forsurvival during nitrogen starvation stress” Plant Cell, 16:1564-1574,2004. Mycelia, collected from 2-day-old liquid CM-grown cultures wereused to isolate nucleic acids. The M. grisea strains were cultured for aweek on solid CM to assess the growth and colony characteristics unlessindicated otherwise. Mycelia used for total protein extraction wereobtained by growing the relevant strains in liquid CM for 3 days at 28°C. Mating analyses were performed as described by Valent et al.,“Magnaporthe grisea genes for pathogenicity and virulence identifiedthrough a series of backcrosses” Genetics 127:87-101, 1991.

Conidia were obtained by scraping 8 day old cultures grown underconstant illumination. Mycelial bits were separated from conidia byfiltration through two layers of Miracloth™ (Calbiochem™, San Diego,Calif.) and resuspended to a concentration of 10⁵ spores per milliliterin sterile distilled water. For light microscopy, individual droplets(30 μl) of conidial suspensions were applied to plastic cover slips orother membranes and incubated under humid conditions at roomtemperature. Microscopic observations were made after 3, 6, 12, 24, and36 hours.

cDNA synthesis and subsequent PCR amplification was conducted usingReverse Transcriptase, AMV and Taq DNA polymerase. For the 5′ RACE,Terminal Deoxynucleotidyl Transferase Recombinant (Promega™) was used.The following primers were used for 5′ RACE and 3′ RACE: Abc-5′ RACE5′-CGGAACCAGATATGAGGAGCTGTTGA-3′ (SEQ ID NO:3) and RACE-Oligo dT-anchor5′-GACCACGCGTATCGATGTCGACTTTTTTTTTTTTTTTTC-3′ (SEQ ID NO:4); RACE-anchor5′-GACCACGCGTATCGATGTCGAC-3′ (SEQ ID NO:5), and Abc-3′ RACE5′-TGATGCTGGTGTTGTACATG-3′ (SEQ ID NO:6). For semi-quantitative RT-PCRexperiments, the following primers were used: MgChap1F(CAAGCCGATAGACACACTGAC; SEQ ID NO:7) and MgChap1R (GAACTAATCGTCCTCAGTGC;SEQ ID NO:8) (this ORF is not annotated but maps to contigs 2.1212 and2.1211); MgTubF (AAACAACTGGGCCAAGGGTCACTACA; SEQ ID NO:9) and MgTubR(CCGATGAAAGTCGACGACATCTTGAG; SEQ ID NO:10) corresponding to ORFMG00604.4; and Abc3F (CGTTATGCAGTCCATGTGGATGAGTC; SEQ ID NO:11) andAbc3R (ACGATTTGTTCGCTGCGCACGTCGAT; SEQ ID NO:12). The oligonucleotideprimers for the RT-PCR experiments on hosts were: HvPr5F(CGCAGAGCAACAACAGTAAAGC; SEQ ID NO:13) and HvPr5R (ACGCCTATTATTGGTTGGCG;SEQ ID NO:14); and Hvact10F (CTGTCTTTCCCAGCATTGTA; SEQ ID NO:15) andHvact10R (ATCCTCGGTGCGACACGGAG; SEQ ID NO:16).

For real-time PCR, total RNA was extracted from the requisite samples asdescribed (Ramos-Pamplona and Naqvi, “Host invasion during rice-blastdisease requires carnitine-dependent transport of peroxisomalacetyl-CoA” Mol. Microbiol. 61(1):61-75, 2006), treated with RNAase-freeDNase (Roche Diagnostics™), and 1 μg was reverse-transcribed for 60 minat 42° C. using a Reverse Transcription Kit (Roche Diagnostics™) in thepresence of random primers and oligo (dT₁₈). Quantitative PCR wasperformed by monitoring in real time the increase in fluorescence of theSYBR Green dye on an Applied Biosystems™ Real-Time PCR 7900HTinstrument, according to the manufacturer's instructions. Primers usedfor the amplifications were Pr-1F (ATGGAGGTATCCAAGCTGGCCATT; SEQ IDNO:17); Pr-1R (GTAAGGCCTCTGTCCGACGAAGTT; SEQ ID NO:18); ActinF(TAGTATTGTTGGCCGTCCTCGCCACAC; SEQ ID NO:19); ActinR(GATGGAATGATGAAGCGCATATCCTTC; SEQ ID NO:20); HvPr5F(CGCAGAGCAACAACAGTAAAGC; SEQ ID NO:21); HvPr5R (ACGCCTATTATTGGTTGGCG;SEQ ID NO:22); Hvact10F (CTGTCTTTCCCAGCATTGTA; SEQ ID NO:23) andHvact10R (ATCCTCGGTGCGACACGGAG; SEQ ID NO:24). Each RT-PCRquantification was carried out in triplicate. Melting curve analysis wasapplied to all final PCR products after the cycling protocols. Valuesfor each gene were normalized to the expression level of the respectivecontrol condition and used further to calculate the ratio of theexpression levels of the requisite transcripts (for example,Pr-1/Actin).

Protein purification and immunoblotting procedures were performed asdescribed by Soundararajan et al., “Woronin body function in Magnaporthegrisea is essential for efficient pathogenesis and for survival duringnitrogen starvation stress.” Plant Cell, 16:1564-1574, 2004. Proteinconcentrations were determined according to the Bradford method using acommercial kit (Bio-Rad Laboratories™). The enhanced chemiluminescentmethod (ECL kit; Amersham Biosciences™) was used for Southern andwestern blot analysis.

Multiple sequence alignments were initially performed using ClustalW(Thompson et al., 1994) for the following MDR sequences related to Abc3protein: Mus (ABCB1, NP_(—)035206), Rattus (ABCB1a, NP_(—)596892),Cricetulus (ABCB1, P21448), Homo (ABCB1, AAW82430), Canis (MDR1,AAY67840), Ovis (MDR1, NP_(—)001009790), Xenopus (MDR11, NP_(—)989254),Gallus (LOC420606, XP_(—)418707), Oryza (XP_(—)467259), Chaetomium(CHGG_(—)08744; EAQ84730), Neurospora (re-annotated NCU06011.2 andNCU9975.1), Magnaporthe (ABC3, AAZ81480 and MG09931.4), Fusarium(FG06881.1; FG03323.1; FG08823.1; FG02786.1; FG01684.1), Aspergillus(AAD43626; Afu7g00480; AN9342.2; AN2349.2; XP_(—)747768; BAE64667;BAE61398; AAD25925; AAB88658; AN3608.2; XP 751944), Coccidioides(EAS34680; EAS30718; EAS35426), Saccharomyces (STE6; CAA82054);Schizosaccharomyces (MAM1, P78966; PMD1, CAA20363); Ustilago(UM06009.1), Cryptococcus (CNA07730), Filobasidiella (MDR1, AAC49890),Yarrowia (YALIOB12188g), Leptosphaeria (AAS92552), Venturia (AAL57243),Trichophyton (AAG01549) and Phaeosphaeria (EAT87032).

Phylip 3.6a (Felsenstein, “PHYLIP (phylogeny inference package). Version3.2.” Cladistics, 5:164-166, 1989) was subsequently used, after removingthe gaps from the initial alignment, to create a phylogram representingthe most parsimonious phylogenetic relatedness of Magnaporthe Abc3p withthe other MDR sequences from the indicated genera within the eukaryotes.Bootstrap analysis (500 replicates/iterations) was used to generate thephylogenetic tree using the Neighbor-joining algorithm in Phylip 3.6a.Percent bootstrap support values exceeding 50% were consideredsignificant. The MDR hits related to ABC3 protein were identifiedinitially using the B-Link webtool on the NCBI network. See FIG. 1.

FIG. 2 is a ClustalW diagram (Thompson et al., “CLUSTAL W:improving thesensitivity of progressive multiple sequence alignment through sequenceweighting, positions-specific gap penalties and weight matrix choice”Nucl. Acids Res., 22:4673-4680, 1994) and Phylip 3.6a-assisteddendrogram (Felsenstein, “PHYLIP (phylogeny inference package). Version3.2” Cladistics, 5:164-166, 1989) depicting the most parsimoniousphylogenetic relatedness of Magnaporthe ABC3 protein with the MDRproteins from the indicated genera within eukaryotes. Bootstrapping (500replicates/iterations) was used to generate the phylogenetic tree usingthe Neighbor-joining algorithm in Phylip 3.6a. Percent bootstrap supportfor each clade (when >50%) is indicated below the branch. B-Link webtoolon the NCBI network was initially used to select 44 MDR hits related toABC3 protein.

Comparison of the ABC3 protein sequence with related sequences inSWISS-PROT and other protein sequence resources identified severalmembers of the P-glycoprotein family of ABC transporters. See FIG. 3.The phylogenetic relationship was established using these sequences(FIG. 2). Individual sequence comparisons and phylogenetic analysesbased on CLUSTAL W (FIG. 1), MEGA (Kumar et al., “MEGA 3: integratedsoftware for molecular evolutionary genetics analysis and sequencealignment” Briefings in Bioinformatics, 5:150-163, 2004) and Phylip 3.6arevealed that ABC3 protein likely defines a separate family of fungalMDR transporters distinct from the Pmd1 or the ATRC family (see FIG. 2,arrowheads). This phylogram also showed that ABC3 protein had divergedsignificantly from its most related Step 6 protein in Saccharomyces.There was however a paralogous transporter (MG09931.4; 56% similarity)encoded in Magnaporthe.

In the Clustal W-based amino acid sequence alignment of yeast sequences(FIG. 3), neither ABC3 protein nor MG09931.4 showed any significantsimilarity (average 7% similarity and 4% identity) to the ABC1 (Urban etal., 1999) or ABC3 transporter (Lee et al., 2005) reported inMagnaporthe. ABC3 showed the highest similarity to SNOG_(—)05968 fromPhaeosphaeria (62% similarity and 43% identity) and was found to beclosely related to AtrC from A. nidulans (34% identity and 53%similarity), the fission yeast Pmd1 (35% identity and 54% similarity;Nishi et al., “A leptomycin B resistance gene of Schizosaccharomycespombe encodes a protein similar to the mammalian P-glycoproteins” Mol.Microbiol. 6:761-769, 1992), and the budding yeast Step 6 (Saccharomycesceriviseae) (28% identity and 46% similarity; Ketchum et al., “The yeasta-factor transporter Stelop, a member of the ABC superfamily, couplesATP hydrolysis to pheromone expert” J. Biol. Chem., 27: 29007-29001,2001), whereas ABC1 (M. grisea) showed a higher level of similarity tothe ScPdr5 (S. ceriviseae) and CaCDR1 (Candida albicans) transportersfrom yeasts. See FIG. 3.

Agrobacterium tumefaciens T-DNA-transfer was performed in M. griseausing hygromycin resistance (encoded by hygromycin phosphotransferasegene HPH) as a selectable marker. Copy number of the integron wasidentified by Southern blots using standard procedures as described inSambrook et al., “Molecular Cloning: A Laboratory Manual” Cold SpringHarbor, N.Y.: Cold Spring Harbor Laboratory Press, 1989. Mutant strainsof interest were collected and purified by backcross analysis, progenytesting, random ascospore analysis and by monoconidial isolation. Thesestrategies have been described in detail in the art and were performedas described by Soundararajan et al., “Woronin body function inMagnaporthe grisea is essential for efficient pathogenesis and forsurvival during nitrogen starvation stress” Plant Cell, 16:1564-1574,2004.

TMT2807 was obtained in the above screen as a single-copy insertionalmutant that produced slightly larger appressoria and showed total lossof pathogenicity towards barley explants. DNA sequences flanking theright and left border of the T-DNA insertion in TMT2807 were amplifiedusing the standard TAIL PCR method (Liu et al., “Efficient isolation andmapping of Arabidopsis thaliana T-DNA insert junctions by thermalasymmetric interlaced PCR” Plant J., 8:457-463, 1995) and subsequentlyconfirmed by nucleotide sequencing. A mutant strain carrying the samedisruption of ABC3 as confirmed in TMT2807 was created in a Guy11background and characterized in parallel.

The mutant strain (TMT2807) of M. grisea (wild-type strain Guy11) wasidentified by its dramatic and complete inability to infect the barleycultivar Express™ in an Agrobacterium Transfer-DNA based randominsertional mutagenesis screen for non-pathogenic mutants ofMagnaporthe. See FIGS. 4 and 5, which show barley leaf explantsinoculated with conidiospores of TMT2807 or wild type (WT) strain. Theleaf tissue was assessed for disease symptoms after 9 days. Furthermonoconidial and random ascospore-analysis-assisted purification of thisstrain showed that loss of the pathogenesis ability co-segregated withhygromycin resistance, which was conferred by the single copyintegration of the HPH1-containing transfer DNA in this strain.

Standard molecular manipulations were performed according to standardmethods. See Sambrook et al., “Molecular Cloning: A Laboratory Manual”Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press, 1989.Fungal genomic DNA was extracted with the potassium acetate method asdescribed by Naqvi et al., “Identification of RAPD markers linked to amajor gene for blast resistance in rice” Mol. Breed., 1:341-348, 1995.Plasmid DNA was isolated using Qiagen™ plasmid preparation kits.Nucleotide sequencing was performed using the ABI Prism big dyeterminator method. Homology searches of DNA/protein sequences wereperformed using the BLAST™ program according to the methods described byAltschul et al., “Gapped BLAST and PSI-BLAST: A new generation ofprotein database search programs” Nucleic Acids Res. 25:3389-3402, 1997.GeneWise™-based predictions were made using the public domain databaseof the European Bioinformatics Institute. Total RNA was isolated withthe RNeasy™ Plant Mini kit (Qiagen™) and cDNA synthesis and subsequentPCR amplification was conducted using Reverse Transcriptase, AMV (RocheDiagnostics™, and Taq DNA Polymerase (Roche™).

Thermal asymmetric interlaced (TAIL) PCR-based sequence analysis (Liu etal., “Efficient isolation and mapping of Arabidopsis thaliana T-DNAinsert junctions by thermal asymmetric interlaced PCR” Plant J.8:457-463, 1995) revealed that the T-DNA insertion in TMT2807 haddisrupted the genomic region corresponding to Contig 67 on Supercontig5.117 (Magnaporthe Genome Database, Release 5, Broad Institute, USA).Further subcloning and sequence analysis showed that the T-DNA insertionin TMT2807 disrupted a region just proximal (231 bp upstream) to exon1of open reading frame MGG_(—)13762.5 (FIG. 6) and led to a total loss oftranscription of this ORF. Analysis of a BAC clone 22C21 (identified byusing the T-DNA insertion flanks from TMT2807 as probes) revealed thepresence of the ORF mentioned above as a 6.3 kilo-basepair (kb) HindIIIfragment. See FIG. 6. This gene was designated abc3 because itspredicted product showed high degree of sequence similarity to theATP-binding cassettes (ABC) encoded by the genes belonging to the ABCtransporter superfamily.

FIG. 6 is a schematic representation of the annotated abc3 locusspanning a HindIII-PvuII fragment from Contig 67 on Supercontig 5.117 inM. grisea. Solid bars and short open boxes indicate the coding regionsand introns respectively and are drawn to scale. RB and LB represent theright and left border sequences of T-DNA (open box) integrated in themutant strain. Opposing arrows demarcate the genomic region deleted inthe abc3Δ strain, that was created using flanking homology (dashedlines) based gene replacement with the hygromycin-resistance cassette(HPH1). Restriction enzyme sites HindIII (H) and PvuII (P) have beendepicted. The scale bar corresponds to 1 kb and also denotes the probeused for Southern analysis shown in FIG. 7.

Genomic DNA from wild-type (WT), abc3Δ, Complemented strain (Comp; abc3Δcarrying an ectopic single-copy integration of the HindIII fragmentdescribed above) and TMT2807 strain was digested with HindIII and probedwith the 1 kb ABC3 fragment or the HPH1-specific fragment. See FIG. 7.The appearance of the 3.2 kb band in the abc3Δ strain, with theconcomitant loss of the wild-type 6.3 kb ABC3 locus, was diagnostic ofthe correct gene replacement event. Ectopic integration of the rescueconstruct in the complemented strain resulted in the retention of the3.2 kb fragment and the restoration of the 6.3 kb band. The presence ofa 0.6 kb fragment in the TMT2807 strain was due to an internal HindIIIsite at the right-border end of the integrated T-DNA in TMT2807. Thisintegron also accounts for the ˜7 kb fragment (the abc3 gene disruptedwith the HPH1 T-DNA cassette) detected in this mutant. A DNA gel blotwith HPH1 as the probe further confirmed the identity of the fragments.Molecular size markers in kilo-basepair are indicated. See FIG. 7.

A full-length abc3 cDNA was obtained by 3′ and 5′ rapid amplification ofthe cDNA ends (RACE). The abc3 locus spanned nucleotides 124832 to131097 on Supercontig 177. The abc3 cDNA sequence was confirmed byobtaining and sequencing several RT-PCR fragments representing theoverall abc3 coding sequence. In each instance, DNA sequence analysiswas carried out on both strands of at least 2 individual clones.Subsequent analysis revealed the existence of 13 exons in the abc3 openreading frame as opposed to the autocall-predicted eleven exons inMGG_(—)13762.5. In addition, 3 nucleotide changes were uncovered in exon3 of MGG_(—)13762.5. Typical regulatory elements related to fungalpromoter regions preceded the abc3 open reading frame. Based on the cDNAsequence, the abc3 gene was predicted to encode a 1321 amino-acidprotein (ABC3) composed of two homologous halves, each with sixmembrane-spanning segments (ABC transmembrane domain) and an ABC ATPasemotif. ABC3 protein thus showed an overall structure and domainorganization typical of the Cluster II.2 (or TC#3.A.1.201) typeP-glycoprotein MDR transporters (Decottignies and Goffeau, “Completeinventory of the yeast ABC proteins” Nat. Genet., 15:137-145, 1997;Saier and Paulsen, “Phylogeny of multidrug transporters” Semin. CellDev. Biol., 12:205-213, 2001).

The complete nucleotide sequence and annotation details for abc3 havebeen deposited in GenBank under accession number DQ156556 thedisclosures of which are hereby incorporated by reference. The sequenceof an abc3 coding region is provided as SEQ ID NO:1 (FIG. 8) and an ABC3protein as SEQ ID NO:2 (FIG. 9). See Table I, below, for the finalannotation of the protein, based on the abc3 Rescue HinDIII fragment andcDNA. Nucleic acids which encode the protein of SEQ ID NO:2 also formpart of the present invention, as well as any nucleic acid which encodesa protein having substantially the same activity of SEQ ID NO:2. An ABC3protein includes any protein which is native to a Magnaporthe species,is at least 95% homologous to SEQ ID NO:2, and which, if not present ornot active, results in loss or substantial reduction (>90%) ofappressorial function and plant pathogenicity. An abc3 gene a usedherein is a nucleic acid that encodes an ABC3 protein.

TABLE I Final Annotation of ABC3. From To Length (bp) Exon 1 1040 112081 Intron 1 1121 1173 53 Exon 2 1174 1422 249 Intron 2 1423 1469 47 Exon3 1470 1974 505 Intron 3 1975 2022 48 Exon 4 2023 2119 97 Intron 4 21202166 47 Exon 5 2167 2530 364 Intron 5 2530 2575 45 Exon 6 2576 2830 255Intron 6 2831 2882 52 Exon 7 2883 3612 730 Intron 7 3613 3662 50 Exon 83663 4365 703 Intron 8 4366 4415 50 Exon 9 4416 4465 50 Intron 9 44664514 49 Exon 10 4515 4540 26 Intron 10 4541 4591 51 Exon 11 4592 4631 40Intron 11 4632 4682 51 Exon 12 4683 5521 839 Intron 12 5522 5577 56 Exon13 5578 5603 27

Example 2 Deletion of the ABC3 Open Reading Frame

One-step PCR-based gene deletion (using the Ura4+ marker) was performedaccording to the methods of Bahler et al., “Heterologous modules forefficient and versatile PCR-based gene targeting in Schizosaccharomycespombe” Yeast 14:943-951, 1998, using 80 bp flanking sequence homology.Deletion of the pmd1 open reading frame was carried out in the YNB38strain. Stable transformants from EMM minus Uracil medium were tested bycolony PCR. The M. grisea abc3 locus was obtained as a 6.3 Kb HinDIIIfragment from BAC 22C21 and cloned into the unique HinDIII site inpJK148 to obtain pFGLr1, carrying the Leu1+ marker. Vector pFGLr1 waslinearized with EcoRI and transformed by electroporation into the S.pombe pmd1 deletion strain described above. Stable integration of FGLr1was confirmed by PCR analysis and copy number ascertained by Southernblot analysis.

Using plasmid vector pFGLabcKO in a one-step gene replacement technique,an abc3 deletion mutant (hereafter referred as abc3Δ) was created byreplacing the entire 4.61 kb coding region of the abc3 locus with theHPH1 cassette encoding the hygromycin phosphotransferase function.Hygromycin-resistant transformants (HPH1⁺) derived from the wild typeGuy11 strain, carrying single-copy insertion of the replacementcassette, were identified and the desired gene replacement event(abc3::HPH1) confirmed by Southern blotting (FIG. 7). Forcomplementation analysis, a full-length genomic copy of abc3 (FIG. 6;6.3 kb HindIII fragment) was introduced into the abc3Δ strain as asingle ectopic integron. At least two independent strains from eachbackground were examined to assess all the vegetative, reproductive andpathogenesis related defects reported here.

Example 3 Phenotypic Characterization of abc3Δ Strains

pFGLr2 (carrying the full-length abc3 gene) or pBarKS (control) wasintroduced into the abc3Δ strain and the TMT2807 mutant. About 20bialaphos-resistant transformants were screened by DNA gel blot analysisin each instance. Two strains that carried single-copy integration ofthe abc3 gene at an ectopic site in each background (TMT2807 and abc3Δ)were used for further tests.

For abc3 gene deletion and rescue of the abc3Δ mutant, DNA fragmentsabout 1.1 kb each, representing the 5′ and 3′ UTR of abc3 gene, wereobtained by PCR. The fragments were ligated sequentially so as to flankthe hph cassette in pFGL44 to obtain plasmid vector pFGLabcKO. pFGLabcKOwas transformed into M. grisea for replacement of the abc3 gene. Thecomplete M. grisea abc3 locus was obtained as a 6.2 Kb HinDIII fragmentfrom the BAC 22C21 and cloned into the HinDIII site of pFGL97 to obtainpFGLr2 with resistance to bialaphos or ammonium gluphosinate as a fungalselectable marker for the rescue transformation. Southern blot analyseswere performed to confirm successful gene deletion strains andsingle-copy genomic integration of the abc3 rescuing cassette.

Wild-type, abc3Δ mutant and an abc3Δ strain complemented with fulllength ABC3 (Comp) (mycelial plugs or conidial suspension) were grown oncomplete agar medium for a week and photographed. See FIG. 10. Thegrowth characteristics are representative of three independentassessments (total n=27 colonies per strain; P<0.05). When inoculated asmycelial plugs or as a conidial suspension on solidified complete medium(CM), the abc3Δ mutant grew slower (˜30% reduction in colony size,P<0.05) than the wild type Guy11 (FIG. 10), although no apparentdifference in conidiogenesis or the overall colony morphology wasuncovered. Quantitation of data from the material in FIG. 10 is providedgraphically in FIG. 11.

FIGS. 10 and 11 show growth characteristics and mating in abc3Δ mutant,photographically and quantitatively. Perithecia development by theindicated Magnaporthe strains in individual sexual crosses with thetester strain of the opposite mating type, were assessed 3 weeks postinoculation. Mean values (±SD) represent the number of perithecial beaksobserved per mating mix on oatmeal agar medium. Quantitations representthree independent experiments covering a total of 30 crosses per strain.WT refers to the cross between Guy11 (mat1-2) and TH3 (mat1-1). Whencrossed to TH3 (mat1-1), the abc3Δ (mat1-2) mutant showed a slightreduction in its sexual reproduction, displaying a decrease in the totalnumber of perithecia produced per sexual cross. See FIG. 11.

There were no significant difference in conidiation of these moderatelystunted abc3Δ mutant colonies as compared to Guy11. Conidia germinationand appressoria formation were not observably different from wild typein the abc3Δ strain. Such defects were not mating-type specific asjudged by analyzing the sexual crosses between an abc3Δ (mat1-1) andwild type Guy11 (mat1-2) (FIG. 11) and could be attributed to reducedfemale fertility in the Magnaporthe strains used (Valent et al.,“Magnaporthe grisea genes for pathogenicity and virulence identifiedthrough a series of backcrosses” Genetics, 127:87-101, 1991). Thecomplemented abc3Δ strain behaved like the wild type in the ability toform perithecia. This suggested a divergence of function for ABC3protein compared to the related Step 6 protein, which is requiredspecifically for the efflux of only the a-factor pheromone inSaccharomyces. These results indicated that in Magnaporthe, the ABC3function is required for proper vegetative growth of the mycelia, but isdispensable for sexual reproduction.

The germination efficiency and growth of the abc3Δ conidia wereassessed. Conidia produced by the abc3Δ strain were normal in quantity,morphology and germination compared to wild type conidia. See FIG. 12.FIG. 13 shows that upon germination, the abc3Δ conidia producedappressoria (average diameter 11.2±0.7 μm; n=3000; P<0.05) that weremarginally larger than those of the wild type strain (10.4±0.6 μm;n=3000; P<0.05).

Example 4 The Role of ABC3 in Magnaporthe pathogenesis

To assess the role of ABC3 protein during host-associated growth anddevelopment, infection assays were performed on the seedlings of ricecultivars C039 or NIL127 and barley cultivar Express™, testing thepathogenicity of abc3Δ conidia. Blast infection assays on host tissuewere conducted as follows. Rice seedlings belonging to CO39 (compatible)or NIL127 (incompatible) genotypes were spray-inoculated with conidialsuspensions (in 0.02% gelatin in water) from wild-type, abc3Δ or abc3Δmutant strains complemented by the introduction of ABC3 (COMP). Diseasesymptoms were assessed after 10 days.

The abc3Δ mutant (like the TMT2807 strain), displayed a total loss ofpathogenicity and failed to elicit any visible blast symptoms/lesions onthe compatible (CO39) or incompatible (NIL127) rice varieties, whencompared with equivalent spray-inoculations of the wild type Guy11conidia or the conidia from the Complemented strain. See FIG. 14. Underthe same conditions, the wild type and the Complemented strain (COMP)caused typical spindle-like, gray centered and severe blast lesions thatmerged into one another on the inoculated rice leaves (FIG. 14).

Twenty bialaphos-resistant abc3Δ strain and TMT2807 mutant transformantswere produced. The transformants contained pFGLr2 (carrying thefull-length ABC3 gene) or pBarKS (control) then were screened by DNA gelblot analysis. Two strains that carried single-copy integration of theabc3 gene at an ectopic site in each background (TMT2807 and abc3Δ) wereused to analyze the suppression of the various phenotypic defectsobserved in the abc3Δ mutant. The mild reduction in vegetative growthand mating efficiency observed in the abc3Δ strain (and in TMT2807) wascompletely suppressed upon introduction of the wild type ABC3 gene. SeeFIG. 14, COMP].

Introduction of the ABC3 gene also restored the strain's ability topenetrate the host and cause blast disease. See FIG. 14, COMP. Thiscomplemented (COMP) strain was found to be as virulent as the wild typeisolate, when spray-inoculated on rice seedlings. In contrast, thevector control could not suppress these abnormalities (data not shown).Since all the defects in the abc3Δ mutant (and TMT2807) could becompletely restored by reintroduction of the wild type ABC3 allele, weconclude that the phenotypic changes and functional abnormalitiesobserved in the abc3Δ strain resulted solely from the disruption of ABC3function.

The host-defense associated Hypersensitive Reaction (HR) was quantitatedin the challenged seedlings based on real time RT-PCR methodology toderive the ratio of rice Pr-1 to Actin gene expression as described byGilbert et al., “A P-type ATPase required for rice blast disease andinduction of host resistance” Nature, 440:535-539, 2006. The valuesgiven in FIG. 14 are hypersensitive-reaction (HR) assessed after 48hours and represent the ratio of rice Pr-1 gene expression to that ofthe Actin transcript. As shown in FIG. 14, the abc3Δ mutant failed toelicit proper HR during a compatible or incompatible interaction withthe host.

Barley-leaf explants challenged with wild type or abc3Δ mutant for 72hours, were co-stained with 3,3′-diaminobenzidine and Trypan blue inorder to visualize the hypersensitivity reaction symptoms in thechallenged tissue. Wild type strain caused massive damage and cell deathand accumulation of reactive oxygen intermediates in the host leaves,whereas the abc3Δ mutant failed to elicit any visible hypersensitivityreaction symptoms in the host and was devoid of such cell death andaccumulation of reactive oxygen species, except in rare instances (<1%).See FIG. 15 (the arrowhead indicates a rare hypersensitivity reactionevent in the host upon challenge with the mutant).

In order to further confirm the failure to elicit hypersensitivityreaction, induction of Pathogenesis-related protein 5 (Pr-5, GenbankAccession HVU276225) in barley leaves challenged for 48 hours wastested. The ratio of Pr-5 expression to that of a gene with ahouse-keeping function (Actin10-4, Genbank accession HVU21907) was usedto estimate the level of hypersensitivity reaction induction. In mockinoculations and in barley leaves challenged with the abc3Δ mutant, thisratio (Pr-5/HvAct10) averaged 1.4±0.3 over three replicates (P<0.05),whereas the ratio was consistently higher and averaged 2.6±0.5 (P<0.005)when estimated in plants infected by the wild type strain. The abc3Δmutant could still be recovered as viable colony forming units from thechallenged leaves but only up to 30 hours post inoculation. The aboveplant infection data indicates that ABC3 protein is required forMagnaporthe pathogenicity. Upon loss of ABC3 function, the blast fungusis incapable of establishing disease and eventually fails to surviveinside the host.

Since concentrated suspensions of abc3Δ conidia failed to elicit anyhost lesions after surface inoculation, these conidia were subjected totwo additional tests: (1) inoculation on abraded host leaves and (2)direct injection of conidia into the leaf nodes or rice leaf sheaths.Equal numbers of conidia from the wild-type or abc3Δ mutant wereinoculated either on abraded barley leaves (FIG. 16, upper panels) orinjected at the base of barley leaves (FIG. 16, central panels) or thebase of rice-leaf sheaths (FIG. 16, bottom panels). Host damage anddisease lesion formation were subsequently assessed after 10 days.Hypersensitivity reaction was assessed after 48 hours as describedabove. Mock refers to the same inoculation treatments but using a 0.02%gelatin solution devoid of conidiospores.

These assays showed that abc3Δ mutant is incapable of causingdisease-related host damage even when forcibly introduced throughwounded tissue or injected directly into the host plants (FIG. 15).Appropriate positive and negative controls were included and are shownalongside in these assays. Even under conditions that bypass therequirement of appressorium function, the abc3Δ mutant failed to elicitproper hypersensitivity reaction as determined by the ratio ofPr-1/Actin gene expression (FIG. 15).

Example 5 Appressorium Function and Host Penetration Defects in theabc3Δ Strain

Equivalent numbers of conidia from the wild type or abc3Δ mutant wereinoculated onto synthetic membranes (PUDO-193 cellophane) and assessedfor germination, appressoria formation, surface penetration and invasivegrowth. The mutant appressoria were functional on PUDO-193 cellophane(clergeot et al., “PLS1, a gene encoding a tetraspanin-like protein, isrequired for penetration of rice leaf by the fungal pathogen Magnaporthegrisel,” Proc. Natl. Acad. Sci. USA, 98:6963-6968, 2001) and penetratedit (FIG. 17 lower panels, see arrowheads) albeit with a much lowerefficiency (39±2.5% compared to 58±2.1%; P<0.005) than the wild typeappressoria. Invasive growth within the cellophane appeared to becompromised in the abc3Δ strain. Taken together, these data indicatethat the abc3Δ mutant is incapable of eliciting proper hypersensitivityreaction or causing blast disease in the host plants, but althoughinefficiently, is still capable of breaching and invading cellophanemembranes.

To address the question whether the ability to breach the artificialmembranes, but not the host tissue, was due to a decrease inappressorial turgor in the abc3Δ strain, cytorrhysis assays wereperformed according to published methods to estimate the appressorialturgor through the incipient. See Howard et al., “Penetration of hardsubstrates by a fungus employing enormous turgor pressures.” Proc. Natl.Acad. Sci. USA, 88:11281-11284 1991; de Jong et al., “Glycerol generatesturgor in rice blast.” Nature, 389:244-245 1997). Briefly, theappressoria were allowed to form for 45 hours on GelBond™ membrane priorto the addition of the indicated concentration of external glycerol.These appressorial collapse assays revealed that compared to thewild-type, the abc3Δ mutant displayed a slightly lower appressorialturgor which was not statistically significant. See Table II, below.This reduction in turgor in the mutant appressoria was particularlyevident when the external glycerol addition was in the 3.5 to 4.5 molarrange. At lower molar concentrations (1-3 M), the internal turgor levelswere inferred to be equivalent to those estimated in the wild typeappressoria. As judged by light microscopy and by thin-sectiontransmission electron microscopy, melanin deposition appeared similar towild type in the abc3Δ appressoria.

TABLE II Appressorial cytorrhysis assay in wild-type Guy11 and the abc3Δmutant. External Glycerol (Molar Guy11 abc3Δ concentration) %cytorrhysis* % cytorrhysis* 1.0  5.0 ± 1.1  6.2 ± 1.5 2.0  8.2 ± 0.910.0 ± 1.7 3.0 32.0 ± 0.8 33.0 ± 1.0 3.5 63.0 ± 1.1 68.1 ± 1.4 4.0 81.5± 1.7 85.1 ± 1.2 4.5 85.0 ± 0.8 89.2 ± 1.1 *Refers to the mean (±SD) ofthree independent estimations each involving 10³ appressoria (P < 0.05).

The TMT2807 and the abc3Δ strain had been isolated as totallynonpathogenic mutants incapable of causing blast disease or host damage.Therefore, detailed microscopic analyses of the infection cycle wasperformed to determine which stage of the pathogenesis process wascompromised in the abc3Δ strain. Equal numbers of conidia from the wildtype strain or the abc3Δ mutant were inoculated on barley-leaf explants,onion epidermal strips or rice-leaf sheaths and allowed to proceedthrough infection-related development for the indicated time. Theappressoria were assessed for appressorium formation at 24 hours andappressorium function (host penetration and invasion, as judged byaniline blue staining of resultant callose deposits) at 48 hours. Theresulting data are shown in FIG. 18 (onion epidermal strips) and FIG. 19(barley leaf explants), and are quantitated in FIG. 20 (wild type,shaded; abc3Δ mutant, black). The papillary callose deposits (FIG. 19,arrowhead; see FIG. 20 for quantitation) and infection hyphae (FIG. 19,asterisk) were visualized after staining the barley-leaf explants withaniline blue at 48 hours (FIG. 19, top panels) or 72 hours (FIG. 19,lower panels) post inoculation. The inset in FIG. 19 shows a rarepapillary callose deposit (arrowhead) produced by the abc3Δ appressoria.Values in FIG. 20 are the mean±SD from three replicates of theexperiment each involving 10³ conidia.

A striking difference was that compared to the wild type organism, onlya negligible percentage of abc3Δ appressoria (0.7±0.9%; n=3000, overthree replicates) were able to produce penetration pegs as judged byaniline blue staining for papillary callose deposits and infectionhyphae formation. See FIGS. 18, 19 and 20 (48 hours). In the wild typestrain, the ability to form penetration pegs on barley leaves was about83% (±2.9; n=3000) at the equivalent 48-hour time-point. Even uponextended incubation, host surface penetration failed to increase. SeeFIG. 18 (72 hours). Even after 96 hours post inoculation, a vastmajority of the abc3Δ appressoria (−99%) still failed to enter the hostand to elicit callose deposition. In contrast, wild type infectioushyphae within the host were already in their ramifying and proliferatingstage in planta.

To further validate these findings, thin-section transmission electronmicroscopy ultrastructural analysis was performed. Barley leaves werechallenged with conidia from the wild type or the abc3Δ mutant andprocessed for thin-section transmission electron microscopy at theindicated time points post inoculation. Near-median transmissionelectron microscopy sections were selected for assessment and arerepresented in FIG. 21. In each panel of FIG. 21, hcw indicates the hostcell wall. Arrowheads indicate the non-functional penetration pegselaborated by abc3Δ appressoria. Callose deposits are marked by anasterisk. This analysis confirmed that the majority of the abc3Δappressoria failed to penetrate the host surface and did not elicit anyvisible reaction from the plant. See FIG. 21, abc3Δ, 48 hours. Futileattempts at penetration were noticeable in only two appressoria out of142 appressoria sectioned in the abc3Δ mutant. See FIG. 21, abc3Δ, 72hours. In each instance, dense papillary callose deposits obstructingthe site of failed entry were clearly visible. On the other hand, thetransmission electron microscopy sections for wild type appressoriashowed successful penetration and elaboration of typical fungalinfectious hyphae in the host tissue.

Conidia from the wild type or the abc3Δ mutant were inoculated onBarley-leaf explants, onion epidermal strips or rice-leaf sheaths andprocessed for aniline blue staining after 72 hours. See FIG. 22.Arrowheads indicate the infection hyphae within the leaf sheath tissue.Data for the infection hyphae from three independent experiments in eachinstance are graphically represented in FIG. 23, where values (±SD) areindicated as percentage points. These results lead to the conclusionthat a defect in appressorium-mediated host penetration leads to theinability of mutants lacking the ABC3 function to produce pathogenicity.Taken together, these data indicate that Abc3p plays an extremelyimportant role in the host penetration step of disease establishment andprobably also is required for the in planta spreading of the blastfungus.

In addition, abc3Δ organisms were incapable of surviving the hostenvironment, since viable abc3Δ cultures could not be recovered from thepenetration assays or the wounding assays beyond 30 hours postinoculation. Conidia from the wild type (FIG. 24, shaded bars) or theabc3Δ (FIG. 24, black bars) strains were inoculated on barley-leaves(host) either by surface inoculation or the injection method orcellophane membrane and allowed to undergo pathogenic development. Cellviability was quantified at the time-points indicated in FIG. 24 bystaining with Phloxine B. Values represent the mean±SD from threeexperiments. Quantitative cell-viability assays using Phloxine B (whichaccumulates in dead cells) showed that the abc3Δ mutant was incapable ofsurviving the environment encountered within the host irrespective ofthe mode of entry (surface inoculation or injection). See FIG. 24.

Appressorial assays on cellophane revealed that viability and invasivegrowth is significantly compromised in the mutant (FIG. 24, 48 hours;P<0.05). Moreover, adding barley-leaf extract during the appressorialassays on cellophane caused a further and marked reduction in theviability of the mutant therein. See FIG. 24 (48 h+Extr).

For preparing barley-leaf extracts, fresh samples from 21-day-oldseedlings were surface-sterilized and ground to a fine powder usingliquid nitrogen in a mortar and stirred in cold, sterilized distilledwater. Ten milliliters of water per gram of leaf material was added tothe sample. The mixture was kept in a refrigerator for 2 hours and thenstirred on a rotary shaker for 1 hour and centrifuged at 5000 rpm for 15minutes. The supernatant was collected and stored in a refrigeratoruntil it was used as a crude water-soluble extract.

The wild type strain remained largely unperturbed under theseconditions, therefore it is likely that the ABC3 protein plays acritical role during early stages of disease establishment and is mostlikely required for the in planta viability and survival of the blastfungus. A parallel experiment included viability counts duringappressorium development and penetration of cellophane in the presenceof water-soluble extracts from barley leaves for 48 hours (Extr).

Example 6 ABC3 Protein P-Glycoprotein and Multidrug Resistance

Earlier studies have documented the role of P-glycoproteins in theefflux of a diverse range of compounds such as steroid hormones (Uhr etal., “Penetration of endogenous steroid hormones corticosterone,cortisol, aldosterone and progesterone into the brain is enhanced inmice deficient for both mdr1a and mdr1b P-glycoproteins” J.Neuroendocrinol. 14:753-759, 2002), mating pheromone (Ketchum et al.,“The yeast a-factor transporter Step 6p, a member of the ABCsuperfamily, couples ATP hydrolysis to pheromone export” J. Biol. Chem.276:29007-29011, 2001), drugs and antibiotics (Nishi et al., “Aleptomycin B resistance gene of Schizosaccharomyces pombe encodes aprotein similar to the mammalian P-glycoproteins” Mol. Microbiol.6(6):761-769, 1992), and extrusion of ions or solutes (Kimura et al.,“Effects of P-glycoprotein inhibitors on transepithelial transport ofcadmium in cultured renal epithelial cells, LLC-PK1 and LLC-GA5-COL150”Toxicology, 208:123-132, 2005). To test whether ABC3 protein servessimilar extrusion function(s), the drug sensitivity of the abc3Δ strainand wild type Guy11 were investigated with respect to the effect ofmetabolic poisons, antifungal agents, and antibiotics.

Drug sensitivity of cells was assayed by measuring the minimuminhibitory concentration (MIC) of that compound required for inhibitingmycelial growth in wild type Guy11. The compounds used in the drugsensitivity assays were as follows, Valinomycin, Verapamil, Benomyl,Gramicidin D, Brefeldin A, Leptomycin B, Actinomycin D, Fluconazole andItraconazole. For the assay, the cells were cultured on agar mediumplates containing the specified concentration of each drug at 28° C. for2-8 days. For tests on conidial function during pathogenic phase, thespecified amounts of the compounds in the appropriate solvent were addeddirectly to the aqueous suspension of conidia. The sensitivity to eachindicated compound (drug sensitivity) is shown in Table III, below.Among the drugs tested, the abc3Δ strain showed increased sensitivity toValinomycin and Actinomycin D. Multidrug resistance correlated with thefunction of an ABC or P-glycoprotein transporter since it could bereversed by the presence of Verapamil in Guy11. The MIC assays did notshow any difference in the sensitivity of Guy11 and abc3Δ mutant for theother compounds tested such as Brefeldin A, Gramicidin D, Leptomycin B,Benomyl and some azole fungicides.

TABLE III Drug Sensitivity* of Wild-type Strain Guy11 and abc3Δ Mutant.Compound Guy11 abc3Δ Valinomycin 20 μg/ml 3 μg/ml Valinomycin +Verapamil 10 μg/ml 3 μg/ml Actinomycin D 15 μg/ml 3.5 μg/ml ActinomycinD + Verapamil 8 μg/ml 3.5 μg/ml Benomyl 2 μg/ml 2 μg/ml Brefeldin A 40μg/ml 40 μg/ml Gramicidin D 180 μg/ml 180 μg/ml Leptomycin B 7.5 ng/ml7.5 ng/ml Fluconazole 2.5 mg/ml 2.5 mg/ml Itraconazole 2 mg/ml 2 mg/ml*The Minimum Inhibitory Concentration (MIC) that is sufficient toinhibit growth of Magnaporthe vegetative mycelia on agar medium.

Having confirmed an important efflux-related role of ABC3 protein duringthe mycelial growth phase of Magnaporthe, further tests were performedto assess whether ABC3 protein is required during the pathogenic phasefor a similar MDR function. Equivalent serial dilutions of wild type orabc3Δ conidial suspensions were cultured in the presence (20 mg/ml) orabsence (0 mg/ml) of Valinomycin on complete growth medium (FIG. 25) for5 days or on artificial inductive membranes (FIG. 26) for 36 hours.Addition of potassium (KCl at 100 mM) failed to reverse the defectsassociated with Valinomycin action on Magnaporthe condiospores andappressoria.

As shown in FIG. 25, the germ tube growth from abc3Δ conidia wasinhibited upon Valinomycin treatment, whereas the germ tube growth inGuy11 could withstand elevated concentration of Valinomycin (20 mg/ml).On proper inductive surfaces, the wild type conidia germinated andformed appressoria in the presence of Valinomycin, albeit at a lowerfrequency (61±2% versus 93±5% for untreated wild type samples). See FIG.26. The appressoria formed under these conditions were alsosubstantially smaller in size. In contrast, the same concentration ofValinomycin had a very severe and dramatic effect on the conidia of theabc3Δ mutant: a complete blockage of conidial germ tube emergence. SeeFIG. 26.

Daniele and Holian, “A potassium ionophore (valinomycin) inhibitslymphocyte proliferation by its effects on the cell membrane” Proc.Natl. Acad. Sci. USA, 73:3599-3602, 1976 have proposed that valinomycinalso can affect the homeostasis of potassium ions across biologicalmembranes. However, exogenous addition of potassium chloride did notsuppress the effect of Valinomycin on either wild type or abc3Δ conidiaand appressoria. Therefore, it is likely that the ABC3 protein serves anessential multidrug-resistance function both during the vegetative andthe pathogenic phase of the rice-blast fungus and that such MDR activityis most likely towards compounds structurally or functionally similar tovalinomycin.

Fission yeast strains belonging to the relevant genotypes (as specifiedin FIG. 27) were cultured on YES medium in the absence or presence ofthe indicated amounts of Valinomycin. Results were documented 5 dayspost inoculation. Strain D+vector refers to the pmd1D mutant carrying anempty plasmid vector, whereas D+ABC3⁺ denotes the pmd1D mutantexpressing an integrated copy of the ABC3 gene. Thus, ABC3 protein alsocould functionally replace its ortholog Pmd1p from fission yeastsignifying the evolutionary conservation and importance of suchmultidrug resistance phenomenon.

Example 7 ABC3 Protein and Sensitivity to Cellular Stress

High potassium levels could not reverse the effect of Valinomycin. TheABC3 protein may function through some as yet unknown mechanism tomodulate its MDR activity towards such antifungal compounds. To testwhether ABC3 is required for modulating osmotic, oxidative and relatedcellular stress conditions, further studies were performed. Mycelialplugs (FIG. 28) or equivalent serial dilutions of conidiosporesuspensions (FIG. 29) from abc3Δ or wild type strains were cultured onminimal agar medium containing the indicated amounts of hydrogenperoxide. The results were documented after an incubation period of oneweek.

Compared to the wild type strain, abc3Δ mutant did not show anydifference in its mycelial growth in the presence of high concentrationsof osmolytes such as sorbitol, sucrose or sodium chloride. In contrast,the abc3Δ mutant was found to be highly sensitive to oxidative stress.Even relatively low concentrations of hydrogen peroxide (2 mM) werelethal to the mutant strain (FIG. 28), whereas the wild type Guy11strain was unaffected under these adverse growth conditions (FIG. 28).The abc3Δ mutant also showed similar sensitivity to paraquat andmenadione, which stimulate accumulation of reactive oxygenspecies/intermediates. The sensitivity of abc3Δ mutant to such oxidativestress was common to the vegetative as well as the pathogenic growthphase (FIG. 29).

FIG. 30 shows nitroblue tetrazolium (NBT)-assisted visualization of theaccumulation of reactive oxygen radicals in wild type and abc3Δcolonies. Three-(top panels) or six-(lower panels) day-old wild type orabc3Δ colonies were stained with NBT solution to detect peroxideaccumulation. The mutant hyphae accumulated relatively higher amounts ofperoxide and likely a higher amount of reactive oxygen species. In FIG.30, the dark centers in the wild type colonies are due to the enhancedcontrast of the image.

Accumulation of hydrogen peroxide was visualized with diaminobenzidine(DAB) in wild type and in abc3Δ mutant appressoria formed on artificialmembranes. Wild type and abc3Δ appressoria formed on artificialmembranes (upper panels) or on barley leaf explants (lower panels) werestained with diaminobenzidine to detect the accumulation of hydrogenperoxide (arrowheads). Arrows indicate the excessdiaminobenzidine-reactive deposits within the appressorial lumen. SeeFIG. 31, upper panels. This showed that the mutant accumulates arelatively higher amount of peroxide levels, which likely would lead toa higher amount of reactive oxygen species. Increased accumulation ofperoxides/DAB-positive material also was detected in the abc3Δ mutantappressoria on artificial membranes and barley-leaf explants (FIG. 31,lower panels).

Fluorimetric evaluation of hydrogen peroxide levels in wild type andabc3Δ strains on barley leaves, after staining with the dye CM-DCFDA(chloromethyl-2′,7′-dichlorofluorescein diacetate, Molecular Probes,USA), revealed that the mutant appressoria accrue 3.5 fold higheramounts of ROS/peroxide than those estimated in the wild-type (FIG. 32).Total extracts from wild type or abc3Δ appressoria were treated withCM-DCFDA and processed for fluorimetric estimations. Values in the graphrepresent fold change (mean±SD; from three independent experiments) inthe CM-DCFDA-reactive material from mutant appressorial extractscompared to those in the wild type. Average value for the CM-DCFDAestimates in the wild type extracts was set at one. An earlier study hasshown that oxidative stress (accumulation of DAB-positive material)precedes the elicitation of cell death in the interacting epidermalcells and the underlying mesophyll cells in Brachypodium infected withMagnaporthe (Routledge et al., “Magnaporthe grisea interactions with themodel grass Brachypodium distachyon closely resemble those with rice(Oryza sativa)” Mol. Plant Path., 5:253-265, 2005). These resultsindicate that ABC3 protein protects the blast fungus' from oxidativedamage in the mycelial growth phase and also during the host-associatedstage of pathogenic development and proliferation.

Example 8 Partial Suppression of abc3Δ Defects, and Effect of Verapamiland Oxidative Stress

To understand whether the pathogenicity defects in abc3Δ are in any wayrelated to the excess build-up of H₂O₂ therein, attempts were made toreduce the levels of peroxide oxidants during appressorium developmentby using several agents such as, Diphenyleneiodonium (DPI; 15 mm),nordihydroguaiaretic acid (NDGA, 10 mm), 5 mm Rotenone (Chiarugi et al.,2003) or antioxidants (Ascorbate or N-acetylcysteine).

Antioxidant treatment partially restored appressorium function in abc3Δwild type (FIG. 33, shaded bars) or abc3Δ (FIG. 33, black bars). Theconidia were tested for appressorium formation and function (hostpenetration) on barley-leaf explants in the presence of DPI, ascorbateor N-acetylcysteine. This test also was carried out in the presence ofVerapamil (Verapam), a generic inhibitor of MDR-based efflux functions.The treatments were carried out for 48 hours in each instance and hostpenetration was assessed by aniline blue staining. The addition ofDiphenyleneiodonium suppressed the host penetration defects associatedwith the abc3Δ appressoria in 5.1±1.1% appressoria (n=3000). See FIG.33. Antioxidant treatment with either ascorbate or N-acetylcysteinerestored penetration function in 23±2.5% and 19±1.6% appressoria,respectively (n=3000, P<0.005), as judged by papillary callosedeposition. See FIG. 33. However, the resultant mutant penetration pegsstill failed to elaborate proper infection hyphae and were unable toadvance the invasion further. The reduction of the intracellularperoxide levels (or oxidative stress) in abc3Δ therefore leads to asignificant suppression of the appressorial defects in this mutant.

In a converse experiment, the effect of exogenous hydrogen peroxide,menadione or paraquat on the ability of the wild type appressoria topenetrate the host surfaces was tested. Conidia from the wild-type (FIG.34, shaded bars) or the abc3Δ strain (FIG. 34, black bars) wereinoculated on barley-leaf explants in the presence of either hydrogenperoxide or paraquat or Menadione and the resultant appressoriumformation and function (host penetration) assessed after 48 hours asdescribed above. Values represent mean±SD from three independentexperiments each involving 2000 conidia per strain.

As expected, in the absence of exogenous peroxide, the wild typeappressoria behaved normally and were able to successfully penetrate thehost surface as judged by papillary callose deposits upon aniline bluestaining. The percentage of appressoria that elaborated penetration pegswas 68±2.3% (n=3000, over 3 replicates), after the 48-hour time point.See FIG. 34. Addition of 3 mM peroxide to the wild type conidia caused asignificant decrease in host penetration with only 36±2.8% appressoria(n=3000, over 3 replicates) able to breach the host surface. See FIG.34. Addition of paraquat or Menadione had a slightly reduced effect onhost penetration of the wild type strain, as compared to treatment withperoxide. See FIG. 34. Such oxidative stress caused a significantreduction in the efficiency of appressorium formation and peroxidelevels exceeding 5 mM blocked germ tube emergence in the wild typeconidia.

Downregulation of MDR activity by Verapamil during pathogenicdevelopment produced a significant decrease (maximal 74%) in penetrationpeg formation, but without affecting the germ tube emergence or growth.These results suggest that excessive peroxide levels have an adverseeffect on the host-penetration capacity of Magnaporthe, and thatreducing the intracellular levels of peroxide (or oxidative stress) inabc3Δ significantly suppresses the appressorial defects observed in thismutant. Taken together, it is clear that ABC3 protein performs animportant MDR function during the pathogenic phase and possibly protectsthe blast fungus from peroxide-based internal oxidative stress duringvegetative growth and pathogenic development.

Example 9 Subcellular Localization of GFP-Tagged ABC3 Protein

The predicted secondary structure of ABC3 protein suggests that it is anintegral membrane protein. In order to determine the distributionpattern of ABC3-protein within the various cell types of Magnaporthe,the abc3-GFP fusion construct was introduced into the Guy11 strain. Twotransformants carrying single copy of abc3:GFP allele, tagged at thegenomic locus, and two control strains were identified by PCR analysesand further confirmed by Southern and western blot analyses. GFPdetection was performed either by epifluorescence microscopy or byconfocal laser-scanning fluorescence microscopy. Strains expressingABC3-GFP showed no obvious differences in growth or pathogenesiscompared to the wild type strain, suggesting that ABC3-GFP is fullyfunctional. ABC3-GFP was judged to be a plasma membrane resident proteinbased on the distinct green fluorescence in the mycelial outermembranes. See FIG. 35. No detectable GFP signals were observed in theconidia of the ABC3:GFP strain (FIG. 35), although the germ tubes showeda faint GFP signal in the plasma membrane.

A time-course analysis during appressorium development and maturitysuggested that the ABC3-GFP protein was highly expressed andconcentrated in the appressorial plasma membrane right from theincipient stage onwards (FIG. 36). Appressorium development in the germtubes of the ABC3-GFP strain was monitored over a 24 hour period. At theindicated time points post germination, GFP epifluorescence was imagedto locate the ABC3-GFP fusion protein. At 17 hours post germination,during appressorium maturation, ABC3-GFP signal was predominantly foundin the vacuolar compartments (FIG. 36, 20 hours) as well, although theplasma membrane residency was diminished substantially.

For vacuolar staining, LysoTracker™ Red DND-99 (50 nM finalconcentration) and Neutral Red (50 nM) were used as per themanufacturer's (Invitrogen™) recommendation. Co-localization studies toconfirm vacuolar residency involved staining with LysoTracker Red DND-99or with Neutral Red. Appressoria formed by the Abc3-GFP strain werestained with LysoTracker Red DND-99 (left panel, vacuoles) or withNeutral Red (right panel) at the 20 hour time point to visualizevacuoles. See FIG. 37.

Nitroblue tetrazolium (Sigma-Aldrich™) was used at 4 mg/ml (inde-ionised water) and the staining carried out for 1 hour at roomtemperature prior to observation. Cell viability was measured bystaining dead cells as follows: samples were stained with 20 mg/mlPhloxine B (Sigma-Aldrich, USA) for 1 hour at room temperature and thesamples subjected to a short wash with PBS prior to microscopic analysisand quantification. As controls for dead cells, wild-type cells wereincubated for 30 min at 65° C. and stained with Phloxine B in parallel.The ABC3-GFP localization during host penetration and invasive growthrevealed that ABC3-GFP was present in the plasma membrane of the newlyformed penetration hyphae (FIG. 38) but was undetectable in theresultant infection hyphae. Consistent with its role in appressoriumfunction and its localization pattern described above, these dataindicate that Magnaporthe ABC3 protein is a transmembrane protein foundin the mycelia and predominantly in the appressoria. It therefore ispossible that it is most likely required for the efflux of toxicmetabolite(s), and/or for regulating the oxidative stress within thesehighly important fungal infection structures.

Reactive oxygen species were detected by staining with a solution of 3,3-Diaminobenzidine (DAB, 2 mg/ml) for 2 hours followed by clearing in asolution of acetic acid:glycerol:ethanol (1:2:2) at 80° C. for 5 min andsubsequently stored in 3% glycerol or by staining with2′,7′-dichlorofluorescein diacetate (10 μM; in 30 mM KCl and 10 mMMES-KOH pH 6) for 1 h at room temperature. For fungus-inoculated leafmaterial, the DAB and Trypan blue stainings were essentially asdescribed (Thordol-Christensen et al., 1997 and Gilbert et al., “AP-type ATPase required for rice blast disease and induction of hostresistance” Nature, 440:535-539, 2006 respectively). In the co-stainingmethod, diaminobenzidine was used prior to sample clarification andTrypan blue treatment. Antioxidants ascorbic acid and N-acetylcysteinewere used at 5 mM concentration.

The intracellular formation of reactive oxygen species in appressoriawas quantified using the fluorescent probe CM-DCFDA(chloromethyl-2′,7′-dichlorofluorescein diacetate; InvitrogenCorporation, USA). The appressorial preparation from the requisitestrain was washed two times in phosphate-buffered saline and exposed to50 mM CM-DCFDA in PBS for 1 hour at 28° C. The appressoria then werewashed two times in PBS, solubilized in water, and homogenized in a MiniBead-beater™ to obtain the intracellular extracts. The fluorescence wasdetermined at 488/525 nm, normalized on a protein basis, and expressedas fold change over the wild type control, which was arbitrarily set at1.

At a later stage during the appressorium formation (17 hours postgermination), ABC3-GFP signal was predominantly found in the vacuolarcompartments as well, although the plasma membrane residency was notaltered. See FIG. 39. Consistent with its role in appressorium function,the localization pattern described above indicates that ABC3 protein isa transmembrane protein found predominantly in the mycelia and theappressoria, where it is most likely required for the efflux ofxenobiotic compounds, and/or for regulating the oxidative stress withinthe fungal structures.

Example 10 abc3Δ Appressoria Accumulate Toxic Metabolite(s)

The prediction that ABC3 protein was an MDR protein raised thepossibility that the abc3Δ appressoria could retain cytotoxicmolecule(s) that block the appressorial function of elaboratingpenetration pegs. To investigate this possibility, intracellular andextracellular extracts were prepared from the appressoria of the wildtype or abc3Δ strain as described in Example 9, above. See FIG. 40.These extracts were used individually during plant infections with wildtype conidia by adding the extracts to wild type conidia in barley leafinfection assays at 0 hours (top panels) or 23 hours (bottom panels)post inoculation. A solvent control at the equivalent concentration wasincluded in parallel. The values in FIG. 40 indicate penetrationefficiency (mean values as percentage points±SD from three replicates)calculated after 48 hours by staining papillary callose deposits withaniline blue.

The intracellular extract from the abc3Δ appressoria, when added at thestart of the wild type infection, caused a dramatic (˜74%; P<0.005)reduction in penetration-peg formation, by aniline blue staining forpapillary callose deposits, See FIG. 40. There was no decrease in theappressorium formation capability under these conditions. Thecorresponding extract from the wild type appressoria resulted only in aslight decrease (˜24%) in host penetration. This decrease by the wildtype extract was comparable to the reduction observed when the extractfrom either the wild type or the mutant strain was added after theinfection had proceeded for 23 hours. See FIG. 40. The control extractsdid not cause any significant decrease in the host penetrationcapability of the wild type appressoria, nor did the extracellularextracts from either the wild type or the abc3Δ appressoria.Theoretically, it is possible that the abc3Δ mutant is unable to effluxcertain toxic or inhibitory metabolite(s), the accumulation of which inthe mutant appressoria likely inhibits host penetration.

The ability of the abc3Δ appressoria intracellular neutral toxin extractto suppress penetration of artificial substrates by the wild type ormutant strains was tested. To extract intracellular neutral toxins,about 2×10⁶ conidia (wild type or abc3Δ) were inoculated in de-ionisedwater on the hydrophobic side of GelBond™ membranes (BioWhittakerMolecular Applications™) and allowed to form appressoria for 20 hours atroom temperature under high humidity. The deionized water was aspiratedand the resultant appressoria washed once with chilled water, scrapedimmediately and ground into a fine powder using liquid nitrogen. Thehomogenate was extracted with 1 ml of chloroform/methanol (1/1, v/v) andthe mixture shaken for 10 minutes and centrifuged at 3000 rpm for 5minutes. The extracted material was evaporated to dryness using rotaryevaporation. The residue was partitioned between 1 ml n-hexane and 1 ml90% methanol (1/1, v/v); the n-hexane layer was discarded and themethanol layer evaporated to dryness as above. The solids were thenpartitioned between 1 ml chloroform and 1 ml de-ionized water. Thechloroform layer was extracted with saturated sodium hydrogen carbonatesolution (3×1 ml). The chloroform layer was then concentrated to drynessand contained the neutral extraction (toxins). This was finallyresuspended in 100 ml of deionized water. The same procedure wasrepeated but without any conidia to serve as a solvent-only control forexperiments with the neutral toxins.

To obtain the extracellular toxin(s), about 2×10⁶ conidia (wild type orabc3Δ) were inoculated in deionized water on the hydrophobic side of aGelBond™ film and allowed to form appressoria for 20 hours at roomtemperature under high humidity. All extracellular solution wascollected, centrifuged to pellet the debris, and the remainingsupernatant transferred to fresh tubes and concentrated by drying in anEppendorf™ Concentrator 5301. The dried pellet was resuspended in 0.1 mldeionized water.

Wild type and abc3Δ appressorial assays were performed on PUDO-193cellophane in the presence (+EXT) or absence (−EXT; solvent control) ofthis extract. As shown in FIG. 41, addition of the intracellular neutraltoxin abc3Δ extract caused a significant reduction (˜85% in wild typeand 89% abc3Δ; P<0.005) in appressorium function on cellophanemembranes.

Cell viability was assessed with Phloxine B staining in vegetativemycelia from the wild type (FIG. 42, gray) or abc3Δ (FIG. 42, black) inthe absence (−EXT; solvent control) or presence (+EXT) of theintracellular neutral toxin extract from the abc3Δ appressoria. Theintracellular abc3Δ extract showed minimal effect on the viability ofthe wild type or abc3Δ mycelia.

The intracellular abc3Δ extract caused a dosage-dependent and noticeablereduction in the elaboration of disease symptoms on rice leaves whenincluded during blast infection assays. Wild type conidia were tested inbarley-leaf inoculation assays in the absence (−EXT; solvent control) orpresence (+EXT; at 1× or 0.5× concentration) of the extract from theabc3Δ mutant. A parallel experiment was carried out in the absence ofconidia. Blast symptoms were assessed and documented 5 days postinoculation. The hypersensitivity reaction (mean values±SD of ricePr-1/Actin ratio from three independent experiments; P<0.05) wasestimated at 48 hours after inoculation. See FIG. 43. The mutant extractalso elicited visible hypersensitivity reaction-like symptoms (theoccurrence of brown lesions in the absence of fungal biomass in treatedsamples and the ratio of rice Pr-1 to Actin gene expression). See FIG.43; P<0.05. ABC3 protein therefore is important for the efflux ofcertain appressorial metabolite(s), the accretion of which has anegative effect on host penetration.

Example 11 Regulation of ABC3 Expression

Sequence analysis of the ABC3 promoter did not reveal the presence ofputative cis-acting regulatory elements or enhancer sequences for stressresponse factors. However, given the above findings showing theinvolvement of ABC3 protein in regulating oxidative stress within thefungal cellular structures, tests were performed to determine whethersuch stress conditions directly influenced abc3 expression. Aqualitative and quantitative study of abc3 expression was performedduring the infection-related developmental stages of two independenttransformants, each carrying a single-copy destabilized GFP reporterdriven by the ABC3 promoter.

For GFP fusion with the TrpC terminator, a 1 kb abc3 fragment justproximal to the stop codon and a 1 kb abc3 fragment immediatelydownstream of the stop were ligated to pFGL44 with an HPH marker toobtain pFGLg1. The eGFP segment with the Trpc Terminator was cut out ofpFGL265 by NcoI and PstI double digestion and ligated to pFGLg1 betweenthe two abc3 fragment to obtain pFGLg2. The vector pFGLg2 was introducedinto M. grisea using routine transformation protocols as reported inSoundararajan et al., “Woronin body function in Magnaporthe grisea isessential for efficient pathogenesis and for survival during nitrogenstarvation stress” Plant Cell, 16:1564-1574, 2004. Transformants wereselected on hygromycin and the correct gene replacement event(homology-dependent chromosomal tagging of ABC3 with GFP) confirmed bySouthern and western analysis. The ABC3:GFP strains thus obtained werefurther tested for growth, Valinomycin sensitivity and host-penetrationdefects to ascertain that the ABC3-GFP was not compromised for function.Conidia from the ABC3(p)::GFP strain were allowed to undergoappressorium development for 24 hours in the absence (Control) orpresence (Treated) of 1 mM hydrogen peroxide. See FIG. 44.Epifluorescent microscopic observations were performed at the indicatedtime points to detect GFP expression driven by the abc3 promoter.

GFP epifluorescence was observed using a Zeiss LSM510 inverted confocalmicroscope (Carl Zeiss, NY, USA) equipped with a 30 mW argon laser. Theobjectives were either 63× Plan-Apochromat (numerical aperture 1.4) or a100× Achromat (n.a. 1.25) oil immersion lens. EGFP was imaged with 488nm wavelength laser excitation, using a 505-530 nm band pass emissionfilter, while red fluorescence was detected with 543 nm laser and 560 nmlong pass emission filter. Routine photomicrographs were taken using thePhotometrics™ CoolSNAP-HQ™ camera mounted on a Nikon Eclipse™ 80icompound microscope equipped with differential interference contrastoptics and the standard filters for epifluorescence detection.

As shown in FIG. 44, the destabilized-GFP fluorescence increaseddramatically upon treatment with hydrogen peroxide. The fluorescenceintensification was four fold higher compared to the untreated controlsamples, was maximal after a 9 hour treatment and did not show anychange after the initial increase. See FIG. 44. Low levels of Paraquator Valinomycin elicited a similar increase in the ABC3 promoter drivendestabilized-GFP expression during appressorium development. Theseresults suggest that the upstream regulatory elements of the abc3 generespond to the model molecules that generate reactive oxygen radicalsand that the abc3 transcript likely is regulated in a redox sensitivemanner during infection-related development in Magnaporthe. In aseparate experiment, a 1.8 kb fragment before the translational startsite of ABC3 was used as a promoter for expressing the destabilized-GFPreporter.

To study whether ABC3 expression also is governed by host signals,semi-quantitative RT-PCR was performed to analyze total RNA extracted atvarious time intervals from barley leaves challenged with wild typeconidia, using the methods of Soundararajan et al., “Woronin bodyfunction in Magnaporthe grisea is essential for efficient pathogenesisand for survival during nitrogen starvation stress.” Plant Cell,16:1564-1574, 2004. Along with ABC3, TUB1 (b-Tubulin) and Mg CHAP1 wereincluded as controls. CHAP1 transcript has been shown to be under redoxregulation in Cochliobolus (Lev et al., “Activation of an AP1-LikeTranscription Factor of the Maize Pathogen Cochliobolus heterostrophusin Response to Oxidative Stress and Plant Signal.” Eukaryotic Cell,4:443-454, 2005).

Semi-quantitative RT-PCR-derived products were amplified using ABC3 orMgCHAP1 or MgTUB1 specific primers from total RNA extracted from thewild type strain grown for the indicated hours post inoculation (hpi) onbarley leaves. See FIG. 45. Negative control (−RT) refers to the RNAsample processed without a reverse transcriptase step prior to the PCRamplification. GD refers to the PCR amplification of the respectivefragments from the genomic DNA samples using the corresponding primersets. Molecular mass standards (Kb) are represented in kilo-basepair.

As shown in FIG. 45, the highly abundant TUB1 showed hardly anydifference in its expression during the duration of the assay. ABC3expression was always weaker compared to TUB1, but showed an inductionat 18-20 hours stage post inoculation. Its expression remained stableand strong after the induction at this time-point and continued to be soup to the 36-hour post inoculation (FIG. 45). CHAP1 expression alsopeaked at 12-18 hours but declined slightly after the 24-hour timepoint. Taken together, the results indicate that ABC3 promoter respondspositively to the redox status of the cell and that the ABC3 geneexpression is likely influenced by the host signals as well, with aninduction, just before the fungus readies itself to enter the hosttissue.

Sequences of genes referred to herein are available in the EMBL/Genbankdata libraries under accession numbers DQ 156556 (ABC3), U89895 (Pe-1),AC104285 (Actin), AJ276225 (HvPr5) and U21907 (Hvact10).

REFERENCES

All references cited here and throughout the application are herebyincorporated by reference in their entirety.

-   1. Altschul et al., “Gapped BLAST and PSI-BLAST: A new generation of    protein database search programs.” Nucleic Acids Res., 25:3389-3402,    1997.-   2. Andrade et al., “The ABC transporter AtrB from Aspergillus    nidulans mediates resistance to all major classes of fungicides and    some natural toxic compounds.” Microbiology, 146:1987-1997, 2000a.-   3. Andrade et al., “The role of ABC transporters from Aspergillus    nidulans in protection against cytotoxic agents and in antibiotic    production.” Mol. Gen. Genet., 263:966-977, 2000b.-   4. Bahler et al., “Heterologous modules for efficient and versatile    PCR-based gene targeting in Schizosaccharomyces pombe.” Yeast,    14:943-951, 1998.-   5. Balhadere et al., “Identification of pathogenicity mutants of the    rice blast fungus Magnaporthe grisea by insertional mutagenesis.”    Mol. Plant-Microbe Interact., 12:129-142, 1999.-   6. Bradford, “A rapid and sensitive method for the quantitation of    microgram quantities of protein utilizing the principle of    protein-dye binding.” Anal. Biochem., 72:248-254, 1976.-   7. Chen and Dickman, “Proline suppresses apoptosis in the fungal    pathogen Colletotrichum trifolii.” Proc. Natl. Acad. Sci. USA,    102:3459-3464, 2005.-   8. Chiarugi et al., “Reactive oxygen species as essential mediators    of cell adhesion: the oxidative inhibition of a FAK tyrosine    phosphatase is required for cell adhesion.” J. Cell Biol.,    161:933-944, 2003.-   9. Chida and Sisler, “Restoration of appressorial penetration    ability by melanin precursors in Pyricularia oryzae treated with    anti-penetrants and in melanin-deficient mutants.” J. Pestic. Sci.,    12:49-55, 1987.-   10. Clergeot et al., “PLS1, a gene encoding a tetraspanin-like    protein, is required for penetration of rice leaf by the fungal    pathogen Magnaporthe grisea.” Proc. Natl. Acad. Sci. USA,    98:6963-6968, 2001.-   11. Cole et al., “Overexpression of a transporter gene in a    multidrug-resistant human lung cancer cell line.” Science,    258:1650-1654, 1992.-   12. Daniele and Holian, “A potassium ionophore (valinomycin)    inhibits lymphocyte proliferation by its effects on the cell    membrane.” Proc. Natl. Acad. Sci. USA, 73:3599-3602, 1976.-   13. Decottignies and Goffeau, “Complete inventory of the yeast ABC    proteins.” Nat. Genet., 15:137-145, 1997.-   14. de Jong et al., “Glycerol generates turgor in rice blast.”    Nature, 389:244-245, 1997.-   15. de Waard et al., “Impact of fungal drug transporters on    fungicide sensitivity, multidrug resistance and virulence.” Pest.    Manag. Sci., 62:195-207, 2006.-   16. Dixon et al., “Early events in the activation of plant defense    responses.” Annu. Rev. Phytopathol., 32:479-501, 1994.-   17. Driessen et al., “Diversity of transport mechanisms: common    structural principles.” Trends Biochem. Sci., 25:397-401, 2000.-   18. Felsenstein, “PHYLIP (phylogeny inference package). Version    3.2.” Cladistics, 5:164-166, 1989.-   19. Fleissner et al., “An ATP-binding cassette multidrug-resistance    transporter is necessary for tolerance of Gibberella pulicaris to    phytoalexins and virulence on potato tubers.” Mol. Plant-Microbe    Interact., 15:102-108, 2002.-   20. Gilbert et al., “A P-type ATPase required for rice blast disease    and induction of host resistance.” Nature, 440:535-539, 2006.-   21. Gottesman and Pastan, “Biochemistry of multidrug resistance    mediated by the multidrug transporter.” Annu. Rev. Biochem.,    62:385-427, 1993.-   22. Hamer and Talbot, “Infection-related development in the rice    blast fungus Magnaporthe grisea.” Curr. Opin. Microbiol., 1:693-697,    1998.-   23. Higgins, “ABC transporters: from microorganisms to man.” Annu.    Rev. Cell Biol., 8:67-113, 1992.-   24. Howard et al., “Penetration of hard substrates by a fungus    employing enormous turgor pressures.” Proc. Natl. Acad. Sci. USA,    88:11281-11284, 1991.-   25. Jacobs et al., “An Arabidopsis callose synthase, GSL5, is    required for wound and papillary callose formation.” Plant Cell,    15:2503-2513, 2003.-   26. Ketchum et al., “The yeast a-factor transporter Step 6p, a    member of the ABC superfamily, couples ATP hydrolysis to pheromone    export.” J. Biol. Chem., 276:29007-29011, 2001.-   27. Kimura et al., “Effects of P-glycoprotein inhibitors on    trans-epithelial transport of cadmium in cultured renal epithelial    cells, LLC-PK1 and LLC-GA5-COL150.” Toxicology, 208:123-132, 2005.-   28. Kodama et al., Sakuranetin, a flavonone phytoalexin from    ultraviolet-irradiated rice leaves.” Phytochemistry, 31:3807-3809,    1992.-   29. Kolaczkowski et al., “In vivo characterization of the drug    resistance profile of the major ABC transporters and other    components of the yeast pleiotropic drug resistance network.    Microb.” Drug Resist., 4:143-158, 1998.-   30. Kumar et al., “MEGA 3: Integrated Software for Molecular    Evolutionary Genetics Analysis and Sequence Alignment.” Briefings in    Bioinformatics, 5:150-163, 2004.-   31. Lee et al., “Functional analysis of all nonribosomal peptide    synthetases in Cochliobolus heterostrophus reveals a factor, NPS6,    involved in virulence and resistance to oxidative stress.” Eukaryot.    Cell, 4:545-555, 2005.-   32. Lee et al., “A novel ABC transporter gene ABC2 involved in    multidrug susceptibility but not pathogenicity in rice blast fungus,    Magnaporthe grisea.” Pestic. Biochem. Physiol., 81:13-23, 2005.-   33. Lev et al., “Activation of an AP1-Like Transcription Factor of    the Maize Pathogen Cochliobolus heterostrophus in Response to    Oxidative Stress and Plant Signal.” Eukaryotic Cell, 4:443-454,    2005.-   34. Liu et al., “Efficient isolation and mapping of Arabidopsis    thaliana T-DNA insert junctions by thermal asymmetric interlaced    PCR.” Plant J., 8:457-463, 1995.-   35. Michaelis and Berkower, “Sequence comparison of yeast    ATP-binding cassette proteins.” Cold Spring Harbor Symp Quant. Biol.    LX, 291-307, 1995.-   36. Moreno et al., “Molecular genetic analysis of fission yeast    Schizosaccharomyces pombe.” Methods Enzymol., 194:795-823, 1991.-   37. Naqvi et al., “Identification of RAPD markers linked to a major    gene for blast resistance in rice.” Mol. Breed., 1:341-348, 1995.-   38. Nishi et al., “A leptomycin B resistance gene of    Schizosaccharomyces pombe encodes a protein similar to the mammalian    P-glycoproteins.” Mol. Microbiol., 6:761-769, 1992.-   39. Nomura and Takagi, “Role of the yeast acetyltransferase Mpr1 in    oxidative stress: regulation of oxygen reactive species caused by a    toxic proline catabolism intermediate.” Proc. Natl. Acad. Sci. USA,    101:12616-12621, 2004.-   40. Osbourn, “Preformed Antimicrobial Compounds and Plant Defense    against Fungal Attack.” Plant Cell, 8:1821-1831, 1996.-   41. Ou, “Rice Diseases.” Commonwealth Mycological Institute, Surrey,    1985.-   42. Park et al., “Independent genetic mechanisms mediate turgor    generation and penetration peg formation during plant infection in    the rice blast fungus.” Mol. Microbiol., 53:1695-1707, 2004.-   43. Ramos-Pamplona and Naqvi, “Host invasion during rice-blast    disease requires carnitine-dependent transport of peroxisomal    acetyl-CoA.” Mol. Microbiol., 61(1):61-75, 2006.-   44. Routledge et al., “Magnaporthe grisea interactions with the    model grass Brachypodium distachyon closely resemble those with rice    (Oryza sativa).” Mol. Plant Path., 5:253-265, 2005.-   45. Saier and Paulsen, “Phylogeny of multidrug transporters.” Semin.    Cell Dev. Biol., 12:205-213, 2001.-   46. Sambrook et al., “Molecular Cloning: A Laboratory Manual.” Cold    Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press, 1989.-   47. Soundararajan et al., “Woronin body function in Magnaporthe    grisea is essential for efficient pathogenesis and for survival    during nitrogen starvation stress.” Plant Cell, 16:1564-1574, 2004.-   48. Stergiopoulos et al., “The ABC transporter MgAtr4 is a virulence    factor of Mycosphaerella graminicola that affects colonization of    substomatal cavities in wheat leaves.” Mol. Plant-Microbe Interact.,    16:689-698, 2003.-   49. Talbot, “On the trail of a cereal killer: Exploring the biology    of Magnaporthe grisea.” Annu. Rev. Microbiol., 57:177-202, 2003.-   50. Thompson et al., “CLUSTAL W: improving the sensitivity of    progressive multiple sequence alignment through sequence weighting,    positions-specific gap penalties and weight matrix choice.” Nucl.    Acids Res., 22:4673-4680, 1994.-   51. Tobin et al., “Genes encoding multiple drug resistance-like    proteins in Aspergillus fumigatus and Aspergillus flavus.” Gene,    200:11-23, 1997.-   52. Uhr et al., “Penetration of endogenous steroid hormones    corticosterone, cortisol, aldosterone and progesterone into the    brain is enhanced in mice deficient for both mdr1a and mdr1b    P-glycoproteins.” J. Neuroendocrinol., 14:753-759, 2002.-   53. Urban et al., “An ATP-driven efflux pump is a novel    pathogenicity factor in rice blast disease.” EMBO J., 18:512-521,    1999.-   54. Valent, “Rice blast as a model system for plant pathology.”    Phytopathology, 80:33-36, 1990.-   55. Valent et al., “Magnaporthe grisea genes for pathogenicity and    virulence identified through a series of backcrosses.” Genetics,    127:87-101, 1991.-   56. Veneault-Fourrey et al., “Autophagic fungal cell death is    necessary for infection by the rice blast fungus.” Science,    312:580-583, 2006.-   57. Vermeulen et al., “The ABC transporter BcatrB from Botrytis    cinerea is a determinant of the activity of the phenylpyrrole    fungicide fludioxonil.” Pest Manag. Sci. 57:393-402, 2001.-   58. Vogel and Somerville, “Isolation and characterization of powdery    mildew-resistant Arabidopsis mutants.” Proc. Natl. Acad. Sci. USA,    97:1897-1902, 2000.-   59. Zwiers et al., “ABC transporters of the wheat pathogen    Mycosphaerella graminicola function as protectants against biotic    and xenobiotic toxic compounds.” Mol. Genet. Genomics, 269:499-507,    2003.

1. An isolated nucleic acid encoding the protein-coding region of theabc3 gene.
 2. The nucleic acid of claim 1 which is SEQ ID NO:1.
 3. Thenucleic acid of claim 1 wherein said abc3 gene is selected from thegroup consisting of the abc3 gene of Magnaporthe species, Aspergillusspecies, Ustilago species and Fusarium species.
 4. The ABC3 protein. 5.The ABC3 protein of claim 4 which is selected from the group consistingof the ABC protein of Magnaporthe species, Aspergillus species, Ustilagospecies and Fusarium species.
 6. The ABC protein of claim 4 which is SEQID NO:2.
 7. A nucleic acid that encodes the ABC3 protein of claim
 4. 8.A nucleic acid that encodes the ABC3 protein of claim
 5. 9. A nucleicacid that encodes the ABC protein of claim
 6. 10. A method of renderingplant pathogenic fungal species non-pathogenic to a plant, whichcomprises applying an ABC3 inhibitor to said plant.
 11. A method ofrendering plant pathogenic fungal species non-pathogenic to a plant,which comprises expressing an ABC3 inhibitor in said plant.
 12. Themethod of claim 11 wherein said plant pathogenic fungal species isselected from the group consisting of Magnaporthe species, Aspergillusspecies, Ustilago species and Fusarium species.
 13. The method of claim12 wherein said plant pathogenic fungal species is Magnaporthe species.14. The method of claim 13 wherein said Magnaporthe species isMagnaporthe grisea.
 15. The method of claim 10 wherein said ABC3inhibitor is selected from the group consisting of a peptide, a nucleicacid that encodes said peptide, a protein, a nucleic acid that encodessaid protein, a peptide-mimetic compound, an organic chemical, areplacement plasmid vector, an antisense nucleic acid and anycombination thereof.
 16. The method of claim 11 wherein said ABC3inhibitor is selected from the group consisting of a peptide, a nucleicacid that encodes said peptide, a protein, a nucleic acid that encodessaid protein, a peptide-mimetic compound, an organic chemical, areplacement plasmid vector, an antisense nucleic acid and anycombination thereof.
 17. The method of claim 16 wherein said ABC3inhibitor is pFGLabcKO.
 18. The nucleic acid of claim 1 which is from aplant pathogenic fungus selected from the group consisting ofMagnaporthe species, Aspergillus species, Ustilago species and Fusariumspecies.